Melan-A- carrier conjugates

ABSTRACT

The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a modified virus-like particle (VLP) comprising a VLP which can be loaded with immunostimulatory substances, in particular with DNA oligonucleotides containing non-methylated C and G (CpGs), and particular peptides derived from MelanA linked thereto. Such CpGVLPs are dramatically more immunogenic than their CpG-free counterparts and induce enhanced B and T cell responses. The immune response against MelanA peptide analogues optionally coupled, fused or attached otherwise to the VLPs is similarly enhanced as the immune response against the VLP itself. In addition, the T cell responses against the MelanA peptide analogues are especially directed to the Thl type. Antigens attached to CpG-loaded VLPs may therefore be ideal vaccines for prophylactic or therapeutic vaccination against allergies, tumors and other self-molecules and chronic viral diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of International ApplicationPCT/EP2004/003164, international filing date of Mar. 25, 2004, andclaims the benefit of U.S. Provisional Application No. 60/457,348, filedMar. 26, 2003, which is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the fields of vaccinology,immunology and medicine. The invention provides compositions and methodsfor enhancing immunological responses against MelanA peptide analogueswhich are coupled, fused or attached otherwise to virus-like particles(VLPs) by binding, preferably by packaging immunostimulatory substances,in particular immunostimulatory nucleic acids, and even more particularoligonucleotides containing at least one non-methylated CpG sequence,into the VLPs. The invention can be used to induce strong and sustainedT cell responses particularly useful for the treatment of tumors.

2. Related Art

The essence of the immune system is built on two separate foundationpillars: one is specific or adaptive immunity which is characterized byrelatively slow response-kinetics and the ability to remember; the otheris non-specific or innate immunity exhibiting rapid response-kineticsbut lacking memory.

It is well established that the administration of purified proteinsalone is usually not sufficient to elicit a strong immune response;isolated antigen generally must be given together with helper substancescalled adjuvants. Within these adjuvants, the administered antigen isprotected against rapid degradation, and the adjuvant provides anextended release of a low level of antigen.

Unlike isolated proteins, viruses induce prompt and efficient immuneresponses in the absence of any adjuvants both with and without T-cellhelp (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)).Many viruses exhibit a quasi-crystalline surface that displays a regulararray of epitopes which efficiently crosslinks epitope-specificimmunoglobulins on B cells (Bachmann & Zinkernagel, Immunol. Today17:553-558 (1996)). Viral structure is even linked to the generation ofanti-antibodies in autoimmune disease and as a part of the naturalresponse to pathogens (see Fehr, T., et al., J. Exp. Med. 185:1785-1792(1997)). Thus, antigens on viral particles that are organized in anordered and repetitive array are highly immunogenic since they candirectly activate B cells and induce the generation of a cytotoxic Tcell response, another crucial arm of the immune system.

Viral particles as antigens exhibit two advantages over their isolatedcomponents: (1) due to their highly repetitive surface structure, theyare able to directly activate B cells, leading to high antibody titersand long-lasting B cell memory; and (2) viral particles, but not solubleproteins, have the potential to induce a cytotoxic T cell response, evenif the viruses are non-infectious and adjuvants are absent.

In addition, DNA rich in non-methylated CG motifs (CpG), as present inbacteria and most non-vertebrates, exhibits a potent stimulatoryactivity on B cells, dendritic cells and other APC's in vitro as well asin vivo. Although bacterial DNA is immunostimulatory across manyvertebrate species, the individual CpG motifs may differ. In fact, CpGmotifs that stimulate mouse immune cells may not necessarily stimulatehuman immune cells and vice versa. In addition, immunostimulatoryCpG-oligodeoxynucleotides induce strong side effects by causingextramedullary hemopoiesis accompanied by splenomegaly andlymphadenopathy in mice (Sparwasser et al., J. Immunol. (1999),162:2368-74 and Example 18).

There have been remarkable advances made in vaccination strategiesrecently, yet there remains a need for improvement on existingstrategies. In particular, there remains a need in the art for thedevelopment of new and improved vaccines that promote a strong CTLimmune response and anti-pathogenic protection as efficiently as naturalpathogens in the absence of generalized activation of APCs and othercells.

Melanomas are aggressive, frequently metastatic tumors derived fromeither melanocytes or melanocyte related nevus cells. Melanomas make upapproximately three percent of all skin cancers and the worldwideincrease in melanoma is unsurpassed by any other neoplasm with theexception of lung cancer in women. Even when melanoma is apparentlylocalized to the skin, up to 30% of the patients will develop systemicmetastasis and the majority will die. In the past decade immunotherapyand gene therapy have emerged as new and promising methods for treatingmelanoma, for example, treatment of Melanoma patients with the MelanA/MART-1 peptide with or without adjuvants. These Strategies are usuallyof limited success. Moreover, most of the studies did not measuredirectly the ex vivo CTL response with MHC class I multimers but ratherused CTL cultures and stimulated them for several weeks before theycould eventually measure a MelanA specific CTL response. In general,peptides are not immunogenic by itself and have a very short half life.

Another way of immunotherapy is the loading of dendritic cells witheither the MelanA/MART-1 Peptide, or transfection of dendritic cellswith MelanA/MART-1-RNA and re-injection onto patients. Drawback of thisprocedure is the purification and incubation of autologous dendriticcells from each individual patient for several days with cytokines invitro. This is very delicate because the dendritic cells have to be inthe right state of maturation for being immunogenic and not tolerogenicthat could lead to T cells no responding to the tumor anymore.

Another approach from Dudley, M. E. (Science. 2002 Oct25;298(5594):850-4) the is isolation of MelanA-specific T cells fromautologous tumor-material of patients, in vitro cultivation andexpansion and reinjection into the donor. As the aforementionedapproach, a specific vaccine needs to produced separately for eachindividual patient and is therefore not the most efficient therapy.

Characterization of potent melanoma vaccines is, therefore, importantfor the development of new strategies for cancer immunotherapy, inparticular for melanoma.

SUMMARY OF THE INVENTION

This invention is based on the finding that particular human MelanApeptide analogues when bound to a core particle having a structure withan inherent repetitive organization, and hereby in particular tovirus-like-particles (VLPs) and subunits of VLPs, respectively, whichVLPs are packaged with immunostimulatory substances (ISSs) such as DNAoligonucleotides, represent potent immunogens for the induction ofspecific antibodies. The invention is further based on the finding thatimmunostimulatory substances such as DNA oligonucleotides can bepackaged into VLPs which renders them more immunogenic. Unexpectedly,the nucleic acids and oligonucleotides, respectively, present in VLPscan be replaced specifically by the immunostimulatory substances andDNA-oligonucleotides containing CpG motifs, respectively. Surprisingly,these packaged immunostimulatory substances, in particularimmunostimulatory nucleic acids such as unmethylated CpG-containingoligonucleotides retained their immunostimulatory capacity withoutwidespread activation of the innate immune system. The compositionscomprising VLP's and the immunostimulatory substances in accordance withthe present invention, and in particular the CpG-VLPs are dramaticallymore immunogenic than their CpG-free counterparts and induce enhanced Band T cell responses. In addition, the T cell responses against both theVLPs and MelanA peptide analogues are especially directed to the Th1type. Human MelanA peptide analogues attached to CpG-loaded VLPs maytherefore be ideal vaccines for prophylactic or therapeutic vaccinationagainst tumors.

In a first embodiment, the invention provides a composition, typicallyand preferably for enhancing an immune response in an animal, comprisinga virus-like particle, an immunostimulatory substance, preferably animmunostimulatory nucleic acid, and even more preferably an unmethylatedCpG-containing oligonucleotide, and at least one antigen or antigenicdeterminant, where the immunostimulatory substance, nucleic acid oroligonucleotide is coupled, fused, or otherwise attached to or enclosedby, i.e., bound, to the virus-like particle and wherein said antigen orantigenic determinant is bound to said virus-like particle and whereinsaid antigen comprises, alternatively consists essentially of, oralternatively consists, of a human melanoma MelanA peptide analogue.

In a preferred embodiment of the invention, the immunostimulatorynucleic acids, in particular the unmethylated CpG-containingoligonucleotides are stabilized by phosphorothioate modifications of thephosphate backbone. In another preferred embodiment, theimmunostimulatory nucleic acids, in particular the unmethylatedCpG-containing oligonucleotides are packaged into the VLPs by digestionof RNA within the VLPs and simultaneous addition of the DNAoligonucleotides containing CpGs of choice. In an equally preferredembodiment, the VLPs can be disassembled before they are reassembled inthe presence of CpGs.

In a further preferred embodiment, the immunostimulatory nucleic acidsdo not contain CpG motifs but nevertheless exhibit immunostimulatoryactivities. Such nucleic acids are described in WO 01/22972. Allsequences described therein are hereby incorporated by way of reference.

In a further preferred embodiment, the virus-like particle is arecombinant virus-like particle. Also preferred, the virus-like particleis free of a lipoprotein envelope. Preferably, the recombinantvirus-like particle comprises, or alternatively consists of, recombinantproteins of Hepatitis B virus, BK virus or other human Polyoma virus,measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth-Disease virus,Retrovirus, Norwalk virus or human Papilloma virus, RNA-phages,Qβ-phage, GA-phage, fr-phage and Ty. In a specific embodiment, thevirus-like particle comprises, or alternatively consists of, one or moredifferent Hepatitis B virus core (capsid) proteins (HBcAgs). In afurther preferred embodiment, the virus-like particle comprisesrecombinant proteins, or fragments thereof, of a RNA-phage. PreferredRNA-phages are Qβ-phage, AP 205-phage, GA-phage, fr-phage.

In a particular embodiment, the antigen comprises, or alternativelyconsists of, a cytotoxic T cell epitope. In a related embodiment, thevirus-like particle comprises the Hepatitis B virus core protein and thecytotoxic T cell epitope is fused to the C-terminus of said Hepatitis Bvirus core protein. In one embodiment, they are fused by a leucinelinking sequence. In a particularly preferred embodiment, the antigen isa polypeptide suited to induce an immune response against cancer cells.

In another aspect of the invention, there is provided a method ofenhancing an immune response in a human or other animal speciescomprising introducing into the animal a composition comprising avirus-like particle, an immunostimulatory substance, preferably animmunostimulatory nucleic acid, and even more preferably an unmethylatedCpG-containing oligonucleotide, and at least one antigen or antigenicdeterminant, where the immunostimulatory substance, preferably thenucleic acid, and even more preferably the oligonucleotide is bound(i.e. coupled, attached or enclosed) to the virus-like particle, andwherein said antigen comprises, alternatively consists essentially of,or alternatively consists of a human melanoma MelanA peptide analogue,and wherein said human melanoma MelanA peptide analogue is bound to saidvirus-like particle.

In yet another embodiment of the invention, the composition isintroduced into an animal subcutaneously, intramuscularly, intranasally,intradermally, intravenously or directly into a lymph node. In anequally preferred embodiment, the immune enhancing composition isapplied locally, near a tumor or local viral reservoir against which onewould like to vaccinate.

In a preferred aspect of the invention, the immune response is a T cellresponse, and the T cell response against the antigen is enhanced. In aspecific embodiment, the T cell response is a cytotoxic T cell response,and the cytotoxic T cell response against the MelanA peptide isenhanced.

The present invention also relates to a vaccine comprising animmunologically effective amount of the immune enhancing composition ofthe present invention together with a pharmaceutically acceptablediluent, carrier or excipient. In a preferred embodiment, the vaccinefurther comprises at least one adjuvant. The invention also provides amethod of immunizing and/or treating an animal comprising administeringto the animal an immunologically effective amount of the disclosedvaccine.

In a preferred embodiment of the invention, the immunostimulatorysubstance-containing VLPs, preferably the immunostimulatory nucleicacid-containing VLP's, an even more preferably the unmethylatedCpG-containing oligonucleotide VLPs are used for vaccination of animals,typically and preferably humans, against melanoma, or MelanA peptides,respectively. The modified VLPs can typically and preferably be used tovaccinate against tumors. The vaccination can be for prophylactic ortherapeutic purposes, or both.

The route of injection is preferably subcutaneous or intramuscular, butit would also be possible to apply the CpG-containing VLPsintradermally, intranasally, intravenously or directly into the lymphnode. In an equally preferred embodiment, the CpG-containing MelanApeptide analogue-coupled or free VLPs are applied locally, near a tumoror local viral reservoir against which one would like to vaccinate.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the SDS-PAGE analysis of Qb-MelanA VLPs. MelanA-peptideswere coupled to Qb VLPs, as described in Example 20. The final productswere mixed with sample buffer and separated under reduced conditions on16 % Novex® Tris-Glycine gels for 1.5 hours at 125 V. The separatedproteins were stained by soaking the gel in Coomassie blue solution.Background staining was removed by washing the gel in 50% methanol, 8%acetic acid. The Molecular weight marker (P 77085, New England BioLabs,Beverly, USA) was used as reference for Qb-MelanA migration velocity(lane 1). 14 μg of either Qb alone (lane 2) or Qb derivatized with SMPH(lane 3) were loaded for comparison with 8 μg of each final product:Qb-MelanA 16-35 (lane 4), Qb-MelanA 16-35 A/L (lane 5), Qb- MelanA 26-35(lane 6) and Qb- MelanA 26-35 A/L (lane 7).

FIG. 2A shows IFN alpha released in the supernatants of ISS-treatedhuman PBMC. PBMC were obtained from buffy coat and incubated withfivefold dilution of the indicated ISS for 18 h. The term G10 is usedfor the the oligonucleotide G10-PO, and the term G3 is used for theoligonucleotide G3-6). Supernatants were collected and IFN alpha wasmeasured by ELISA, using a set of antibodies provided by PBL BiomedicalLaboratories.

FIG. 2B shows the upregulation of CD69 on human CD8+ PBMC treated withISS. PBMC were obtained from buffy coat and incubated with fivefolddilution of the indicated ISS for 18 h. Cells were washed and incubatedwith anti-CD8-FITC, anti-CD19-PE and anti-CD69-APC (all from BDPharMingen) for 20 min on ice. After washing, cells were analysed on aFACS Calibur using CellQuest software.

FIG. 3 shows the virus titers after immunizing mice with Qbx33 packagedwith poly (I:C), G3-6, or G6. C57B16 mice were immunized by injectingeither 100 μg Qbx33, 100 μg Qb VLPs packaged with poly (I:C) and coupledto p33 (Qb-pIC-33, also termed QbxZnxpolyICxp33GGC), 90 μg Qbx33packaged with G3-6 (Qbx33/G3-6), or 90 μg Qbx33 packaged with G6(Qbx33/G6). After eight days, mice were challenged with 1.5×106 plaqueforming units Vaccinia virus, carrying the LCMV-p33 epitope. Five dayslater, mice were sacrificed and the ovaries were collected. A singlecell suspension from the ovaries was prepared and added to BCS40 cellsin serial dilutions. One day later, the cell layer was stained with asolution containing 50% Ethanol, 2% formaldehyde, 0.8% NaCl and 0.5%Crystal violet) and the viral plaques were counted.

DETAILED DESCRIPTION OF THE INVETION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are hereinafter described.

1. Definitions

Amino acid linker: An “amino acid linker”, or also just termed “linker”within this specification, as used herein, either associates the antigenor antigenic determinant with the second attachment site, or morepreferably, already comprises or contains the second attachment site,typically—but not necessarily—as one amino acid residue, preferably as acysteine residue. The term “amino acid linker” as used herein, however,does not intend to imply that such an amino acid linker consistsexclusively of amino acid residues, even if an amino acid linkerconsisting of amino acid residues is a preferred embodiment of thepresent invention. The amino acid residues of the amino acid linker are,preferably, composed of naturally occuring amino acids or unnaturalamino acids known in the art, all-L or all-D or mixtures thereof.However, an amino acid linker comprising a molecule with a sulfhydrylgroup or cysteine residue is also encompassed within the invention. Sucha molecule comprise preferably a C1-C6 alkyl-, cycloalkyl (C5, C6), arylor heteroaryl moiety. However, in addition to an amino acid linker, alinker comprising preferably a C1-C6 alkyl-, cycloalkyl- (C5, C6), aryl-or heteroaryl-moiety and devoid of any amino acid(s) shall also beencompassed within the scope of the invention. Association between theantigen or antigenic determinant or optionally the second attachmentsite and the amino acid linker is preferably by way of at least onecovalent bond, more preferably by way of at least one peptide bond.

Animal: As used herein, the term “animal” is meant to include, forexample, humans, sheep, horses, cattle, pigs, dogs, cats, rats, mice,mammals, birds, reptiles, fish, insects and arachnids.

Antibody: As used herein, the term “antibody” refers to molecules whichare capable of binding an epitope or antigenic determinant. The term ismeant to include whole antibodies and antigen-binding fragments thereof,including single-chain antibodies. Most preferably the antibodies arehuman antigen binding antibody fragments and include, but are notlimited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv) and fragmentscomprising either a VL or VH domain. The antibodies can be from anyanimal origin including birds and mammals. Preferably, the antibodiesare human, murine, rabbit, goat, guinea pig, camel, horse or chicken. Asused herein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulins and that do not express endogenousimmunoglobulins, as described, for example, in U.S. Pat. No. 5,939,598by Kucherlapati et al.

Antigen: As used herein, the term “antigen” refers to a molecule capableof being bound by an antibody or a T cell receptor (TCR) if presented byMHC molecules. The term “antigen”, as used herein, also encompassesT-cell epitopes. An antigen is additionally capable of being recognizedby the immune system and/or being capable of inducing a humoral immuneresponse and/or cellular immune response leading to the activation of B-and/or T-lymphocytes. This may, however, require that, at least incertain cases, the antigen contains or is linked to a T helper cellepitope (Th cell epitope) and is given in adjuvant. An antigen can haveone or more epitopes (B- and T-epitopes). The specific reaction referredto above is meant to indicate that the antigen will preferably react,typically in a highly selective manner, with its corresponding antibodyor TCR and not with the multitude of other antibodies or TCRs which maybe evoked by other antigens. Antigens as used herein may also bemixtures of several individual antigens.

A “tumor antigen” as used herein is a compound, such as a peptide,associated with a tumor or cancer and which is capable of provoking animmune response. In particular, the compound is capable of provoking animmune response when presented in the context of an MHC molecule. Tumorantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen, et al.,Cancer Research, 54:1055 (1994), by partially purifying the antigens, byrecombinant technology or by de novo synthesis of known antigens. Tumorantigens include antigens that are antigenic portions of or are a wholetumor or cancer polypeptide. Such antigens can be isolated or preparedrecombinantly or by any other means known in the art. Cancers or tumorsinclude, but are not limited to, biliary tract cancer; brain cancer;breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

Antigenic determinant: As used herein, the term “antigenic determinant”is meant to refer to that portion of an antigen that is specificallyrecognized by either B- or T-lymphocytes. B-lymphocytes respond toforeign antigenic determinants via antibody production, whereasT-lymphocytes are the mediator of cellular immunity. Thus, antigenicdeterminants or epitopes are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors.

Antigen presenting cell: As used herein, the term “antigen presentingcell” is meant to refer to a heterogenous population of leucocytes orbone marrow derived cells which possess an immunostimulatory capacity.For example, these cells are capable of generating peptides bound to MHCmolecules that can be recognized by T cells. The term is synonymous withthe term “accessory cell” and includes, for example, Langerhans' cells,interdigitating cells, B cells, macrophages and dendritic cells. Undersome conditions, epithelial cells, endothelial cells and other, non-bonemarrow derived cells may also serve as antigen presenting cells.

Association: As used herein, the term “association” as it applies to thefirst and second attachment sites, refers to the binding of the firstand second attachment sites that is preferably by way of at least onenon-peptide bond. The nature of the association may be covalent, ionic,hydrophobic, polar or any combination thereof, preferably the nature ofthe association is covalent, and again more preferably the associationis through at least one, preferably one, non-peptide bond. As usedherein, the term “association” as it applies to the first and secondattachment sites, not only encompass the direct binding or associationof the first and second attachment site forming the compositions of theinvention but also, alternatively and preferably, the indirectassociation or binding of the first and second attachment site leadingto the compositions of the invention, and hereby typically andpreferably by using a heterobifunctional cross-linker.

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element of non-natural or natural origin typicallyand preferably being comprised by the virus-like particle, to which thesecond attachment site typically and preferably being comprised by theMelanA peptide analogue of the invention may associate. The firstattachment site may be a protein, a polypeptide, an amino acid, apeptide, a sugar, a polynucleotide, a natural or synthetic polymer, asecondary metabolite or compound (biotin, fluorescein, retinol,digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combinationthereof, or a chemically reactive group thereof. The first attachmentsite is located, typically and preferably on the surface, of thevirus-like particle. Multiple first attachment sites are present on thesurface of virus-like particle typically in a repetitive configuration.Preferably, the first attachment site is an amino acid or a chemicallyreactive group thereof.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element associated with, typically and preferablybeing comprised by, the MelanA peptide analogue of the invention towhich the first attachment site located on the surface of the virus-likeparticle may associate. The second attachment site of the MelanA peptideanalogue of the invention may be a protein, a polypeptide, a peptide, asugar, a polynucleotide, a natural or synthetic polymer, a secondarymetabolite or compound (biotin, fluorescein, retinol, digoxigenin, metalions, phenylmethylsulfonylfluoride), or a combination thereof, or achemically reactive group thereof. At least one second attachment siteis present on the MelanA peptide analogue of the invention. The term“MelanA peptide analogue with at least one second attachment site”refers, therefore, to an antigen or antigenic construct comprising atleast the MelanA peptide analogue of the invention and the secondattachment site. However, in particular for a second attachment site,which is of non-natural origin, i.e. not naturally occurring within theMelanA peptide analogue of the invention, these antigen or antigenicconstructs comprise an “amino acid linker”.

Bound: As used herein, the term “bound” refers to binding that may becovalent, e.g., by chemically coupling, or non-covalent, e.g., ionicinteractions, hydrophobic interactions, hydrogen bonds, etc. Covalentbonds can be, for example, ester, ether, phosphoester, amide, peptide,imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. Theterm “bound” is broader than and includes terms such as “coupled”,“fused”, “associated” and “attached”. Moreover, with respect to theimmunostimulatory substance being bound to the virus-like particle theterm “bound” also includes the enclosement, or partial enclosement, ofthe immunostimulatory substance. Therefore, with respect to theimmunostimulatory substance being bound to the virus-like particle theterm “bound” is broader than and includes terms such as “coupled,”“fused,” “enclosed”, “packaged” and “attached.” For example, theimmunostimulatory substance such as the unmethylated CpG-containingoligonucleotide can be enclosed by the VLP without the existence of anactual binding, neither covalently nor non-covalently.

Coat protein(s): As used herein, the term “coat protein(s)” refers tothe protein(s) of a bacteriophage or a RNA-phage capable of beingincorporated within the capsid assembly of the bacteriophage or theRNA-phage. However, when referring to the specific gene product of thecoat protein gene of RNA-phages the term “CP” is used. For example, thespecific gene product of the coat protein gene of RNA-phage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” of bacteriophage Qβcomprise the “Qβ CP” as well as the A1 protein. The capsid ofBacteriophage Qβ is composed mainly of the Qβ CP, with a minor contentof the A1 protein. Likewise, the VLP Qβ coat protein contains mainly QβCP, with a minor content of A1 protein.

Coupled: As used herein, the term “coupled” refers to attachment bycovalent bonds or by non-covalent interactions. With respect to thecoupling of the antigen to the virus-like particle the term “coupled”preferably refers to attachment by covalent bonds. Moreover, withrespect to the coupling of the antigen to the virus-like particle theterm “coupled” preferably refers to association and attachment,respectively, by at least one non-peptide bond. Any method normally usedby those skilled in the art for the coupling of biologically activematerials can be used in the present invention.

Fusion: As used herein, the term “fusion” refers to the combination ofamino acid sequences of different origin in one polypeptide chain byin-frame combination of their coding nucleotide sequences. The term“fusion” explicitly encompasses internal fusions, i.e., insertion ofsequences of different origin within a polypeptide chain, in addition tofusion to one of its termini.

CpG: As used herein, the term “CpG” refers to an oligonucleotide whichcontains at least one unmethylated cytosine, guanine dinucleotidesequence (e.g. “CpG DNA” or DNA containing a cytosine followed byguanosine and linked by a phosphate bond) and stimulates/activates, e.g.has a mitogenic effect on, or induces or increases cytokine expressionby, a vertebrate cell. For example, CpGs can be useful in activating Bcells, NK cells and antigen-presenting cells, such as monocytes,dendritic cells and macrophages, and T cells. The CpGs can includenucleotide analogs such as analogs containing phosphorothioester bondsand can be double-stranded or single-stranded. Generally,double-stranded molecules are more stable in vivo, while single-strandedmolecules have increased immune activity.

Epitope: As used herein, the term “epitope” refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. An “immunogenicepitope,” as used herein, is defined as a portion of a polypeptide thatelicits an antibody response or induces a T-cell response in an animal,as determined by any method known in the art. (See, for example, Geysenet al., Proc. Natl. Acad. Sci. USA 81:3998 4002 (1983)). The term“antigenic epitope,” as used herein, is defined as a portion of aprotein to which an antibody can immunospecifically bind its antigen asdetermined by any method well known in the art. Immunospecific bindingexcludes non specific binding but does not necessarily exclude crossreactivity with other antigens. Antigenic epitopes need not necessarilybe immunogenic. Antigenic epitopes can also be T-cell epitopes, in whichcase they can be bound immunospecifically by a T-cell receptor withinthe context of an MHC molecule.

An epitope can comprise 3 amino acids in a spatial conformation which isunique to the epitope. Generally, an epitope consists of at least about5 such amino acids, and more usually, consists of at least about 8-10such amino acids. If the epitope is an organic molecule, it may be assmall as Nitrophenyl. Preferred epitopes are the MelanA-peptideanalogues of the invention.

Immune response: As used herein, the term “immune response” refers to ahumoral immune response and/or cellular immune response leading to theactivation or proliferation of B- and/or T-lymphocytes. In someinstances, however, the immune responses may be of low intensity andbecome detectable only when using at least one substance in accordancewith the invention. “Immunogenic” refers to an agent used to stimulatethe immune system of a living organism, so that one or more functions ofthe immune system are increased and directed towards the immunogenicagent. An “immunogenic polypeptide” is a polypeptide that elicits acellular and/or humoral immune response, whether alone or linked to acarrier in the presence or absence of an adjuvant.

Immunization: As used herein, the terms “immunize” or “immunization” orrelated terms refer to conferring the ability to mount a substantialimmune response (comprising antibodies or cellular immunity such aseffector CTL) against a target antigen or epitope. These terms do notrequire that complete immunity be created, but rather that an immuneresponse be produced which is substantially greater than baseline. Forexample, a mammal may be considered to be immunized against a targetantigen if the cellular and/or humoral immune response to the targetantigen occurs following the application of methods of the invention.

Immunostimulatory nucleic acid: As used herein, the termimmunostimulatory nucleic acid refers to a nucleic acid capable ofinducing and/or enhancing an immune response. Immunostimulatory nucleicacids, as used herein, comprise ribonucleic acids and in particulardeoxyribonucleic acids. Preferably, immunostimulatory nucleic acidscontain at least one CpG motif e.g. a CG dinucleotide in which the C isunmethylated. The CG dinucleotide can be part of a palindromic sequenceor can be encompassed within a non-palindromic sequence.Immunostimulatory nucleic acids not containing CpG motifs as describedabove encompass, by way of example, nucleic acids lacking CpGdinucleotides, as well as nucleic acids containing CG motifs with amethylated CG dinucleotide. The term “immunostimulatory nucleic acid” asused herein should also refer to nucleic acids that contain modifiedbases such as 4-bromo-cytosine.

Immunostimulatory substance: As used herein, the term “immunostimulatorysubstance” refers to a substance capable of inducing and/or enhancing animmune response. Immunostimulatory substances, as used herein, include,but are not limited to, toll-like receptor activing substances andsubstances inducing cytokine secretion. Toll-like receptor activatingsubstances include, but are not limited to, immunostimulatory nucleicacids, peptideoglycans, lipopolysaccharides, lipoteichonic acids,imidazoquinoline compounds, flagellins, lipoproteins, andimmunostimulatory organic substances such as taxol.

The term “natural human Melan A peptide” or “normal human Melan Apeptide” as used herein, shall refer to a peptide comprising, oralternatively consisting essentially of, or alternatively consisting ofthe amino acid sequence EAAGIGILTV (SEQ ID NO: 78) representing aminoacids positions 26-35 of the normal human MelanA protein sequence orAAGIGILTV (SEQ ID NO: 79) representing amino acids positions 27-35 ofthe normal human MelanA protein sequence.

The term “MelanA peptide analogue” or “human MelanA peptide analogue” or“human melanoma MelanA peptide analogue” as used herein shall be definedas a peptide in which the amino acid sequence of the correspondingnormal MelanA peptide is altered by at least one amino acid or aminoacid derivative, wherein this alteration may comprise an amino acidsubstitution and/or deletion and/or insertion or a combination thereof.In a preferred embodiment of the present invention, the term “MelanApeptide analogue” as used herein shall be defined as a peptide in whichthe amino acid sequence of the corresponding normal MelanA peptide (SEQID NO: 91) is altered by three, preferably two, and even more preferablyone, amino acid or amino acid derivative, wherein this alteration maycomprise an amino acid substitution and/or deletion and/or insertion ora combination thereof. In a further preferred embodiment of the presentinvention, the term “MelanA peptide analogue” as used herein shall bedefined as a peptide in which the amino acid sequence of thecorresponding normal MelanA peptide is altered by three, preferably two,and even more preferably one, amino acid or amino acid derivative,wherein this alteration may comprise an amino acid substitution and/ordeletion and/or insertion or a combination thereof, and wherein thisalteration is at position 26, 27, 28 and/or 35 of the normal humanMelanA protein sequence (SEQ ID NO: 91), and wherein said alteration ispreferably an amino acid substitution. The terms “MelanA peptideanalogue”, “human MelanA peptide analogue”, “human melanoma MelanApeptide analogue”, and “human melanoma MelanA/MART-1 peptide analogue”are used interchangeably.

Natural origin: As used herein, the term “natural origin” means that thewhole or parts thereof are not synthetic and exist or are produced innature.

Non-natural: As used herein, the term generally means not from nature,more specifically, the term means from the hand of man.

Non-natural origin: As used herein, the term “non-natural origin”generally means synthetic or not from nature; more specifically, theterm means from the hand of man.

Ordered and repetitive antigen or antigenic determinant array: As usedherein, the term “ordered and repetitive antigen or antigenicdeterminant array” generally refers to a repeating pattern of antigen orantigenic determinant, characterized by a typically and preferablyuniform spacial arrangement of the antigens or antigenic determinantswith respect to the core particle and virus-like particle, respectively.In one embodiment of the invention, the repeating pattern may be ageometric pattern. Typical and preferred examples of suitable orderedand repetitive antigen or antigenic determinant arrays are those whichpossess strictly repetitive paracrystalline orders of antigens orantigenic determinants, preferably with spacings of 0.5 to 30nanometers, more preferably 3 to 15 nanometers, even more preferably 3to 8 nanometers.

Oligonucleotide: As used herein, the terms “oligonucleotide” or“oligomer” refer to a nucleic acid sequence comprising 2 or morenucleotides, generally at least about 6 nucleotides to about 100,000nucleotides, preferably about 6 to about 2000 nucleotides, and morepreferably about 6 to about 300 nucleotides, even more preferably about20 to about 300 nucleotides, and even more preferably about 20 to about100 nucleotides. The terms “oligonucleotide” or “oligomer” also refer toa nucleic acid sequence comprising more than 100 to about 2000nucleotides, preferably more than 100 to about 1000 nucleotides, andmore preferably more than 100 to about 500 nucleotides.“Oligonucleotide” also generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Oligonucleotide” includes, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “oligonucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. Further, an oligonucleotide can besynthetic, genomic or recombinant, e.g., λ-DNA, cosmid DNA, artificialbacterial chromosome, yeast artificial chromosome and filamentous phagesuch as M13. In a very preferred embodiment of the present invention,the oligonucleotide is a synthetic oligonucleotide.

The term “oligonucleotide” also includes DNAs or RNAs containing one ormore modified bases and DNAs or RNAs with backbones modified forstability or for other reasons. For example, suitable nucleotidemodifications/analogs include peptide nucleic acid, inosin, tritylatedbases, phosphorothioates, alkylphosphorothioates, 5-nitroindoledeoxyribofuranosyl, 5-methyldeoxycytosine and5,6-dihydro-5,6-dihydroxydeoxythymidine. A variety of modifications havebeen made to DNA and RNA; thus, “oligonucleotide” embraces chemically,enzymatically or metabolically modified forms of polynucleotides astypically found in nature, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells. Other nucleotideanalogs/modifications will be evident to those skilled in the art.

Packaged: The term “packaged” as used herein refers to the state of animmunostimulatory substance, preferably of an immunostimulatory nucleicacid, in relation to the VLP. The term “packaged” as used hereinincludes binding that may be covalent, e.g., by chemically coupling, ornon-covalent, e.g., ionic interactions, hydrophobic interactions,hydrogen bonds, etc. Covalent bonds can be, for example, ester, ether,phosphoester, amide, peptide, imide, carbon-sulfur bonds such asthioether bonds, carbon-phosphorus bonds, and the like. The term alsoincludes the enclosement, or partial enclosement, of a substance. Theterm “packaged” includes terms such as “coupled, “enclosed” and“attached.” For example, the immunostimulatory substance such as theunmethylated CpG-containing oligonucleotide can be enclosed by the VLPwithout the existence of an actual binding, neither covalently nornon-covalently. In preferred embodiments, in particular, ifimmunostimulatory nucleic acids are the immunostimulatory substances,the term “packaged” indicates that the immunostimulatory nucleic acid ina packaged state is not accessible to DNAse dr RNAse hydrolysis. Inpreferred embodiments, the immunostimulatory nucleic acid is packagedinside the VLP capsids, most preferably in a non-covalent manner.

The compositions of the invention can be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human or other animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application.

Peptide: The term “peptide” as used herein, and in particular withrespect to the human melanoma MelanA peptide or the normal human Melan Apeptide, shall refer to a molecule composed of monomers (amino acids),typically and preferably linearly, linked by amide bonds (also known aspeptide bonds). It indicates a molecular chain of amino acids and doesnot refer to a specific length of the product.

Organic molecule: As used herein, the term “organic molecule” refers toany chemical entity of natural or synthetic origin. In particular theterm “organic molecule” as used herein encompasses, for example, anymolecule being a member of the group of nucleotides, lipids,carbohydrates, polysaccharides, lipopolysaccharides, steroids,alkaloids, terpenes and fatty acids, being either of natural orsynthetic origin. In particular, the term “organic molecule” encompassesmolecules such as nicotine, cocaine, heroin or other pharmacologicallyactive molecules contained in drugs of abuse. In general an organicmolecule contains or is modified to contain a chemical functionalityallowing its coupling, binding or other method of attachment to thevirus-like particle in accordance with the invention.

Polypeptide: As used herein, the term “polypeptide” refers to a moleculecomposed of monomers (amino acids) linearly linked by amide bonds (alsoknown as peptide bonds). It indicates a molecular chain of amino acidsand does not refer to a specific length of the product. Thus, peptides,oligopeptides and proteins are included within the definition ofpolypeptide. This term is also intended to refer to post-expressionmodifications of the polypeptide, for example, glycosolations,acetylations, phosphorylations, and the like. A recombinant or derivedpolypeptide is not necessarily translated from a designated nucleic acidsequence. It may also be generated in any manner, including chemicalsynthesis.

A substance which “enhances” an immune response refers to a substance inwhich an immune response is observed that is greater or intensified ordeviated in any way with the addition of the substance when compared tothe same immune response measured without the addition of the substance.

Preferably, a substance which “enhances” an immune response refersherein (i) to a substance in which the frequency of MelanA-specific,preferably natural human MelanA peptide-specific or human melanomaMelanA peptide analogue-specific, T cells increases when compared to thefrequency of MelanA-specific, preferably natural human MelanApeptide-specific or human melanoma MelanA peptide analogue-specific, Tcells measured without the addition of the substance, or (ii) to asubstance in which the functionality of MelanA-specific, preferablynatural human MelanA peptide-specific or human melanoma MelanA peptideanalogue-specific, T cells deviates, preferably improves, when comparedto the functionality of MelanA-specific, preferably natural human MelanApeptide-specific or human melanoma MelanA peptide analogue-specific, Tcells measured without the addition of the substance, or (iii) to asubstance in which the phenotype of MelanA-specific, preferably naturalhuman MelanA peptide-specific or human melanoma MelanA peptideanalogue-specific, T cells deviates such as the resulting T cells arecapable of increased proliferation, reduced apoptosis or more efficienthoming to tumor tissues, when compared to the proliferation, apoptosisor homing to tumor tissues of MelanA-specific, preferably natural humanMelanA peptide-specific or human melanoma MelanA peptideanalogue-specific, T cells measured without the addition of thesubstance.

The frequency of MelanA-specific, preferably natural human MelanApeptide-specific T cells or human melanoma MelanA peptideanalogue-specific, is measured by way of MHC-class I/peptide complexessuch as tetramer or multimer staining, preferably tetramer staining asdescribed in Speiser, DE. et al. Eur J Immunol. 2002, Vol. 32, 731-741,whereas the functionality of MelanA-specific, preferably natural humanMelanA peptide-specific or human melanoma MelanA peptideanalogue-specific, T cells, is measured by way of cytokine release suchas intracellular staining, cytokine capture assay, Elispot, ELISA, andpreferably by Elispot as described in Speiser, D E. et al. Eur JImmunol. 2002, Vol. 32, 731-741. Moreover, the functionality ofMelanA-specific, preferably natural human MelanA peptide-specific orhuman melanoma MelanA peptide analogue-specific, T cells, can also bemeasured by way of measuring MelanA-specific, preferably natural humanMelanA peptide-specific or human melanoma MelanA peptideanalogue-specific, cytolytic CD8+ T cell response in Chromium orEuropium release assay as described in Valmori, D. et al. J. Immunol.1998, 161, 6956-6962. The phenotyping of MelanA-specific, preferablynatural human MelanA peptide-specific or human melanoma MelanA peptideanalogue-specific, T cells is measured by way of using antibodiesagainst cell surface or intracellular proteins such as cell activationmarkers, cell differentiation markers, homing markers, chemokine andcytokine receptors, costimulatory receptors, death receptors, killeractivatory or inhibitory receptors, integrins, expression ofanti-apoptotic proteins and absence of senescence markers and preferablyby way of cell activation markers as described in Speiser, DE. et al.Eur J Immunol. 2002, Vol. 32, 731-741.

Effective Amount: As used herein, the term “effective amount” refers toan amount necessary or sufficient to realize a desired biologic effect.An effective amount of the composition would be the amount that achievesthis selected result, and such an amount could be determined as a matterof routine by a person skilled in the art. For example, an effectiveamount for treating an immune system deficiency could be that amountnecessary to cause activation of the immune system, resulting in thedevelopment of an antigen specific immune response upon exposure toantigen. The term is also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

Self antigen: As used herein, the term “self antigen” refers to proteinsencoded by the host's genome or DNA and products generated by proteinsor RNA encoded by the host's genome or DNA are defined as self.Preferably, the tem “self antigen”, as used herein, refers to proteinsencoded by the human genome or DNA and products generated by proteins orRNA encoded by the human genome or DNA are defined as self. Theinventive compositions, pharmaceutical compositions and vaccinescomprising self antigens are in particular capable of breaking toleranceagainst a self antigen when applied to the host. In this context,“breaking tolerance against a self antigen” shall refer to enhancing animmune response, as defined herein, and preferably enhancing a B or a Tcell response, specific for the self antigen when applying the inventivecompositions, pharmaceutical compositions and vaccines comprising theself antigen to the host. In addition, proteins that result from acombination of two or several self-molecules or that represent afraction of a self-molecule and proteins that have a high homology twoself-molecules as defined above (>95%, preferably >97%, morepreferably >99%) may also be considered self.

Treatment: As used herein, the terms “treatment”, “treat”, “treated” or“treating” refer to prophylaxis and/or therapy. When used with respectto an infectious disease, for example, the term refers to a prophylactictreatment which increases the resistance of a subject to infection witha pathogen or, in other words, decreases the likelihood that the subjectwill become infected with the pathogen or will show signs of illnessattributable to the infection, as well as a treatment after the subjecthas become infected in order to fight the infection, e.g., reduce oreliminate the infection or prevent it from becoming worse.

Vaccine: As used herein, the term “vaccine” refers to a formulationwhich contains the composition of the present invention and which is ina form that is capable of being administered to an animal. Typically,the vaccine comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a host, the vaccine is able to provokean immune response including, but not limited to, the production ofantibodies, cytokines and/or the activation of cytotoxic T cells,antigen presenting cells, helper T cells, dendritic cells and/or othercellular responses.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention. The term “adjuvant”as used herein refers to non-specific stimulators of the immune responseor substances that allow generation of a depot in the host which whencombined with the vaccine of the present invention provide for an evenmore enhanced immune response. A variety of adjuvants can be used.Examples include incomplete Freund's adjuvant, aluminum hydroxide andmodified muramyldipeptide. The term “adjuvant” as used herein alsorefers to typically specific stimulators of the immune response whichwhen combined with the vaccine of the present invention provide for aneven more enhanced and typically specific immune response. Examplesinclude, but limited to, GM-CSF, IL-2, IL-12, IFNα. Further examples arewithin the knowledge of the person skilled in the art.

Virus-like particle: As used herein, the term “virus-like particle”refers to a structure resembling a virus particle but which has not beendemonstrated to be pathogenic. Typically, a virus-like particle inaccordance with the invention does not carry genetic informationencoding for the proteins of the virus-like particle. In general,virus-like particles lack the viral genome and, therefore, arenoninfectious. Also, virus-like particles can often be produced in largequantities by heterologous expression and can be easily purified. Somevirus-like particles may contain nucleic acid distinct from theirgenome. As indicated, a virus-like particle in accordance with theinvention is non replicative and noninfectious since it lacks all orpart of the viral genome, in particular the replicative and infectiouscomponents of the viral genome. A virus-like particle in accordance withthe invention may contain nucleic acid distinct from their genome. Atypical and preferred embodiment of a virus-like particle in accordancewith the present invention is a viral capsid such as the viral capsid ofthe corresponding virus, bacteriophage, or RNA-phage. The terms “viralcapsid” or “capsid”, as interchangeably used herein, refer to amacromolecular assembly composed of viral protein subunits. Typicallyand preferably, the viral protein subunits assemble into a viral capsidand capsid, respectively, having a structure with an inherent repetitiveorganization, wherein said structure is, typically, spherical ortubular. For example, the capsids of RNA-phages or HBcAg's have aspherical form of icosahedral symmetry. The term “capsid-like structure”as used herein, refers to a macromolecular assembly composed of viralprotein subunits resembling the capsid morphology in the above definedsense but deviating from the typical symmetrical assembly whilemaintaining a sufficient degree of order and repetitiveness.

Virus-like particle of a bacteriophage: As used herein, the term“virus-like particle of a bacteriophage” refers to a virus-like particleresembling the structure of a bacteriophage, being non replicative andnoninfectious, and lacking at least the gene or genes encoding for thereplication machinery of the bacteriophage, and typically also lackingthe gene or genes encoding the protein or proteins responsible for viralattachment to or entry into the host. This definition should, however,also encompass virus-like particles of bacteriophages, in which theaforementioned gene or genes are still present but inactive, and,therefore, also leading to non-replicative and noninfectious virus-likeparticles of a bacteriophage.

VLP of RNA phage coat protein: The capsid structure formed from theself-assembly of 180 subunits of RNA phage coat protein and optionallycontaining host RNA is referred to as a “VLP of RNA phage coat protein”.A specific example is the VLP of Qβ coat protein. In this particularcase, the VLP of Qβ coat protein may either be assembled exclusivelyfrom Qβ CP subunits (SEQ ID: No 10) generated by expression of a Qβ CPgene containing, for example, a TAA stop codon precluding any expressionof the longer A1 protein through suppression, see Kozlovska, T. M., etal., Intervirology 39: 9-15 (1996)), or additionally contain A1 proteinsubunits (SEQ ID: No 11) in the capsid assembly. The readthrough processhas a low efficiency and is leading to an only very low amount of A1protein in the VLPs. An extensive number of examples have been performedwith different combinations of ISS packaged and antigen coupled. Nodifferences in the coupling efficiency and the packaging have beenobserved when VLPs of Qβ coat protein assembled exclusively from Qβ CPsubunits or VLPs of Qβ coat protein containing additionally A1 proteinsubunits in the capsids were used. Furthermore, no difference of theimmune response between these QβVLP preparations was observed.Therefore, for the sake of clarity the term “QβVLP” is used throughoutthe description of the examples either for VLPs of Qβ coat proteinassembled exclusively from Qβ CP subunits or VLPs of Qβ coat proteincontaining additionally A1 protein subunits in the capsids.

The term “virus particle” as used herein refers to the morphologicalform of a virus. In some virus types it comprises a genome surrounded bya protein capsid; others have additional structures (e.g., envelopes,tails, etc.).

Non-enveloped viral particles are made up of a proteinaceous capsid thatsurrounds and protects the viral genome. Enveloped viruses also have acapsid structure surrounding the genetic material of the virus but, inaddition, have a lipid bilayer envelope that surrounds the capsid. In apreferred embodiment of the invention, the VLP's are free of alipoprotein envelope or a lipoprotein-containing envelope. In a furtherpreferred embodiment, the VLP's are free of an envelope altogether.

One, a, or an: When the terms “one,” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

As will be clear to those skilled in the art, certain embodiments of theinvention involve the use of recombinant nucleic acid technologies suchas cloning, polymerase chain reaction, the purification of DNA and RNA,the expression of recombinant proteins in prokaryotic and eukaryoticcells, etc. Such methodologies are well known to those skilled in theart and can be conveniently found in published laboratory methodsmanuals (e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997)).Fundamental laboratory techniques for working with tissue culture celllines (Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition,(1998)) and antibody-based technologies (Harlow, E. and Lane, D.,“Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988); Deutscher, M. P., “Guide to ProteinPurification,” Meth. Enzymol. 128, Academic Press San Diego (1990);Scopes, R. K., “Protein Purification Principles and Practice,” 3rd ed.,Springer-Verlag, New York (1994)) are also adequately described in theliterature, all of which are incorporated herein by reference.

2. Compositions and Methods for Enhancing an Immune Response

The disclosed invention provides compositions and methods for enhancingan immune response against one or more antigens in an animal.Compositions of the invention comprise, or alternatively consistessentially of, or alternatively consist of, a virus-like particle, atleast one immunostimulatory substance, preferably an immunostimulatorynucleic acid, and even more preferably an unmethylated CpG-containingoligonucleotide, and at least one antigen or antigenic determinant,wherein the immunostimulatory substance, the immunostimulatory nucleicacid or the oligonucleotide is bound to the virus-like particle, andwherein said antigen or antigenic determinant is bound to saidvirus-like particle, and wherein said antigen comprises, alternativelyconsists essentially of, or alternatively consists of a human melanomaMelanA peptide analogue. Furthermore, the invention conveniently enablesthe practitioner to construct such a composition for various treatmentand/or prophylactic prevention purposes, which include the preventionand/or treatment of cancers, for example.

Virus-like particles in the context of the present application refer tostructures resembling a virus particle but which are not pathogenic. Ingeneral, virus-like particles lack the viral genome and, therefore, arenoninfectious. Also, virus-like particles can be produced in largequantities by heterologous expression and can be easily purified.

In a preferred embodiment, the virus-like particle is a recombinantvirus-like particle. The skilled artisan can produce VLPs usingrecombinant DNA technology and virus coding sequences which are readilyavailable to the public. For example, the coding sequence of a virusenvelope or core protein can be engineered for expression in abaculovirus expression vector using a commercially available baculovirusvector, under the regulatory control of a virus promoter, withappropriate modifications of the sequence to allow functional linkage ofthe coding sequence to the regulatory sequence. The coding sequence of avirus envelope or core protein can also be engineered for expression ina bacterial expression vector, for example.

Examples of VLPs include, but are not limited to, the capsid proteins ofHepatitis B virus, measles virus, Sindbis virus, rotavirus,foot-and-mouth-disease virus, Norwalk virus, the retroviral GAG protein,the retrotransposon Ty protein p1, the surface protein of Hepatitis Bvirus, human papilloma virus, human polyoma virus, BK virus (BKV), RNAphages, Ty, fr-phage, GA-phage, AP 205-phage and, in particular,Qβ-phage.

As will be readily apparent to those skilled in the art, the VLP of theinvention is not limited to any specific form. The particle can besynthesized chemically or through a biological process, which can benatural or non-natural. By way of example, this type of embodimentincludes a virus-like particle or a recombinant form thereof.

In a more specific embodiment, the VLP can comprise, or alternativelyconsist of, recombinant polypeptides of Rotavirus; recombinantpolypeptides of Norwalk virus; recombinant polypeptides of Alphavirus;recombinant proteins which form bacterial pili or pilus like structures;recombinant polypeptides of Foot and Mouth Disease virus; recombinantpolypeptides of measles virus, recombinant polypeptides of Sindbisvirus, recombinant polypeptides of Retrovirus; recombinant polypeptidesof Hepatitis B virus (e.g., a HBcAg); recombinant polypeptides ofTobacco mosaic virus; recombinant polypeptides of Flock House Virus;recombinant polypeptides of human Papillomavirus; recombinantpolypeptides of Polyoma virus and, in particular, recombinantpolypeptides of human Polyoma virus, and in particular recombinantpolypeptides of BK virus; recombinant polypeptides of bacteriophages,recombinant polypeptides of RNA phages; recombinant polypeptides of Ty;recombinant polypeptides of fr-phage, recombinant polypeptides ofGA-phage, recombinant polypeptides of AP 205-phage and, in particular,recombinant polypeptides of Qβ-phage. The virus-like particle canfurther comprise, or alternatively consist of, one or more fragments ofsuch polypeptides, as well as variants of such polypeptides. Variants ofpolypeptides can share, for example, at least 80%, 85%, 90%, 95%, 97%,or 99% identity at the amino acid level with their wild typecounterparts.

In a preferred embodiment, the virus-like particle comprises, consistsessentially of, or alternatively consists of recombinant proteins, orfragments thereof, of a RNA-phage. Preferably, the RNA-phage is selectedfrom the group consisting of a) bacteriophage Qβ; b) bacteriophage R17;c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f)bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i)bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7; and m)bacteriophage AP205.

In another preferred embodiment of the present invention, the virus-likeparticle comprises, or alternatively consists essentially of, oralternatively consists of recombinant proteins, or fragments thereof, ofthe RNA-bacteriophage Qβ or of the RNA-bacteriophage fr or of theRNA-bacteriophage AP205.

In a further preferred embodiment of the present invention, therecombinant proteins comprise, or alternatively consist essentially of,or alternatively consist of coat proteins of RNA phages.

RNA-phage coat proteins forming capsids or VLPs, or fragments of thebacteriophage coat proteins compatible with self-assembly into a capsidor a VLP, are, therefore, further preferred embodiments of the presentinvention. Bacteriophage Qβ coat proteins, for example, can be expressedrecombinantly in E. coli. Further, upon such expression these proteinsspontaneously form capsids. Additionally, these capsids form a structurewith an inherent repetitive organization.

Specific preferred examples of bacteriophage coat proteins which can beused to prepare compositions of the invention include the coat proteinsof RNA bacteriophages such as bacteriophage Qβ (SEQ ID NO:10; PIRDatabase, Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 11;Accession No. AAA16663 referring to Qβ A1 protein), bacteriophage R17(PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO:13; PIRAccession No. VCBPFR), bacteriophage GA (SEQ ID NO: 14; GenBankAccession No. NP-040754), bacteriophage SP (GenBank Accession No.CAA30374 referring to SP CP and Accession No. NP_(—)695026 referring toSP A1 protein), bacteriophage MS2 (PIR Accession No. VCBPM2),bacteriophage M11 (GenBank Accession No. AAC06250), bacteriophage MX1(GenBank Accession No. AAC14699), bacteriophage NL95 (GenBank AccessionNo. AAC14704), bacteriophage f2 (GenBank Accession No. P03611),bacteriophage PP7 (SEQ ID NO: 19), and bacteriophage AP205 (SEQ ID NO:31). Furthermore, the A1 protein of bacteriophage Qβ or C-terminaltruncated forms missing as much as 100, 150 or 180 amino acids from itsC-terminus may be incorporated in a capsid assembly of Qβ coat proteins.Generally, the percentage of QβA1 protein relative to Qβ CP in thecapsid assembly will be limited, in order to ensure capsid formation.Further specific examples of bacteriophage coat proteins are describedin WO 02/056905 on page 45 and 46 incorporated herein by way ofreference. Further preferred virus-like particles of RNA-phages, inparticular of Qβ in accordance of this invention are disclosed in WO02/056905, the disclosure of which is herewith incorporated by referencein its entirety.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively consists essentially of,or alternatively consists of recombinant proteins, or fragments thereof,of a RNA-phage, wherein the recombinant proteins comprise, consistessentially of or alternatively consist of mutant coat proteins of a RNAphage, preferably of mutant coat proteins of the RNA phages mentionedabove. In another preferred embodiment, the mutant coat proteins of theRNA phage have been modified by removal of at least one lysine residueby way of substitution, or by addition of at least one lysine residue byway of substitution; alternatively, the mutant coat proteins of the RNAphage have been modified by deletion of at least one lysine residue, orby addition of at least one lysine residue by way of insertion. Thedeletion, substitution or addition of at least one lysine residue allowsvarying the degree of coupling, i.e. the amount of human melanoma MelanApeptide analogues per subunits of the VLP of the RNA-phages, inparticular, to match and tailor the requirements of the vaccine. In apreferred embodiment of the present invention, on average at least 1.0human melanoma MelanA peptide analogue per subunit are linked to the VLPof the RNA-phage. This value is calculated as an average over all thesubunits or monomers of the VLP of the RNA-phage. In a further preferredembodiment of the present invention, at least 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9 or at least 2.0 human melanoma MelanA peptideanalogues are linked to the VLP of the RNA-phages as being calculated asa coupling average over all the subunits or monomers of the VLP of theRNA-phage.

In another preferred embodiment, the virus-like particle comprises, oralternatively consists essentially of, or alternatively consists ofrecombinant proteins, or fragments thereof, of the RNA-bacteriophage Qβ,wherein the recombinant proteins comprise, or alternatively consistessentially of, or alternatively consist of coat proteins having anamino acid sequence of SEQ ID NO:10, or a mixture of coat proteinshaving amino acid sequences of SEQ ID NO:10 and of SEQ ID NO: 11 ormutants of SEQ ID NO: 11 and wherein the N-terminal methionine ispreferably cleaved.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, consists essentially of or alternativelyconsists of recombinant proteins of Qβ, or fragments thereof, whereinthe recombinant proteins comprise, or alternatively consist essentiallyof, or alternatively consist of mutant Qβ coat proteins. In anotherpreferred embodiment, these mutant coat proteins have been modified byremoval of at least one lysine residue by way of substitution, or byaddition of at least one lysine residue by way of substitution.Alternatively, these mutant coat proteins have been modified by deletionof at least one lysine residue, or by addition of at least one lysineresidue by way of insertion.

Four lysine residues are exposed on the surface of the capsid of Qβ coatprotein. Qβ mutants, for which exposed lysine residues are replaced byarginines can also be used for the present invention. The following Qβcoat protein mutants and mutant Qβ VLPs can, thus, be used in thepractice of the invention: “Qβ-240” (Lys13-Arg; SEQ ID NO:20), “Qβ-243”(Asn 10-Lys; SEQ ID NO:21), “Qβ-250” (Lys 2-Arg, Lys13-Arg; SEQ IDNO:22), “Qβ-251” (SEQ ID NO:23) and “Qβ-259” (Lys 2-Arg, Lys16-Arg; SEQID NO:24). Thus, in further preferred embodiment of the presentinvention, the virus-like particle comprises, consists essentially of oralternatively consists of recombinant proteins of mutant Qβ coatproteins, which comprise proteins having an amino acid sequence selectedfrom the group of a) the amino acid sequence of SEQ ID NO: 20; b) theamino acid sequence of SEQ ID NO:21; c) the amino acid sequence of SEQID NO: 22; d) the amino acid sequence of SEQ ID NO:23; and e) the aminoacid sequence of SEQ ID NO: 24. The construction, expression andpurification of the above indicated Qβ coat proteins, mutant Qβ coatprotein VLPs and capsids, respectively, are disclosed in WO02/056905. Inparticular is hereby referred to Example 18 of above mentionedapplication.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively consists essentially of,or alternatively consists of recombinant proteins of Qβ, or fragmentsthereof, wherein the recombinant proteins comprise, consist essentiallyof or alternatively consist of a mixture of either one of the foregoingQβ mutants and the corresponding A1 protein.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant proteins, or fragments thereof,of RNA-phage AP205.

The AP205 genome consists of a maturation protein, a coat protein, areplicase and two open reading frames not present in related phages; alysis gene and an open reading frame playing a role in the translationof the maturation gene (Klovins, J., et al., J. Gen. Virol. 83: 1523-33(2002)). AP205 coat protein can be expressed from plasmid pAP283-58 (SEQID NO: 30), which is a derivative of pQb10 (Kozlovska, T. M. et al.,Gene 137:133-37 (1993)), and which contains an AP205 ribosomal bindingsite. Alternatively, AP205 coat protein may be cloned into pQb185,downstream of the ribosomal binding site present in the vector. Bothapproaches lead to expression of the protein and formation of capsids asdescribed in WO 04/007538 which is incorporated by reference in itsentirety. Vectors pQb10 and pQb185 are vectors derived from pGEM vector,and expression of the cloned genes in these vectors is controlled by thetrp promoter (Kozlovska, T. M. et al., Gene 137:133-37 (1993)). PlasmidpAP283-58 (SEQ ID NO:30) comprises a putative AP205 ribosomal bindingsite in the following sequence, which is downstream of the XbaI site,and immediately upstream of the ATG start codon of the AP205 coatprotein: tctagaATTTTCTGCGCACCCAT CCCGGGTGGCGCCCAAAGTGAGGAAAATCACatg(bases 77-133 of SEQ ID NO: 30). The vector pQb185 comprises a ShineDelagarno sequence downstream from the XbaI site and upstream of thestart codon (tctagaTTAACCCAACGCGTAGGAGTCAGGCCatg (SEQ ID NO: 61), ShineDelagarno sequence underlined).

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant coat proteins, or fragmentsthereof, of the RNA-phage AP205.

This preferred embodiment of the present invention, thus, comprisesAP205 coat proteins that form capsids. Such proteins are recombinantlyexpressed, or prepared from natural sources. AP205 coat proteinsproduced in bacteria spontaneously form capsids, as evidenced byElectron Microscopy (EM) and immunodiffusion. The structural propertiesof the capsid formed by the AP205 coat protein (SEQ ID NO: 31) and thoseformed by the coat protein of the AP205 RNA phage are nearlyindistinguishable when seen in EM. AP205 VLPs are highly immunogenic,and can be linked with antigens and/or antigenic determinants togenerate vaccine constructs displaying the antigens and/or antigenicdeterminants oriented in a repetitive manner. High titers are elicitedagainst the so displayed antigens showing that bound antigens and/orantigenic determinants are accessible for interacting with antibodymolecules and are immunogenic.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant mutant coat proteins, orfragments thereof, of the RNA-phage AP205.

Assembly-competent mutant forms of AP205 VLPs, including AP205 coatprotein with the subsitution of proline at amino acid 5 to threonine(SEQ ID NO: 32), may also be used in the practice of the invention andleads to a further preferred embodiment of the invention. These VLPs,AP205 VLPs derived from natural sources, or AP205 viral particles, maybe bound to antigens to produce ordered repetitive arrays of theantigens in accordance with the present invention.

AP205 P5-T mutant coat protein can be expressed from plasmid pAP281-32(SEQ ID NO: 33), which is derived directly from pQb185, and whichcontains the mutant AP205 coat protein gene instead of the Qβ coatprotein gene. Vectors for expression of the AP205 coat protein aretransfected into E. coli for expression of the AP205 coat protein.

Methods for expression of the coat protein and the mutant coat protein,respectively, leading to self-assembly into VLPs are described in WO04/007538, which is incorporated by reference in its entirety. SuitableE. coli strains include, but are not limited to, E. coli K802, JM 109,RR1. Suitable vectors and strains and combinations thereof can beidentified by testing expression of the coat protein and mutant coatprotein, respectively, by SDS-PAGE and capsid formation and assembly byoptionally first purifying the capsids by gel filtration andsubsequently testing them in an immunodiffusion assay (Ouchterlony test)or Electron Microscopy (Kozlovska, T. M. et al., Gene 137:133-37(1993)).

AP205 coat proteins expressed from the vectors pAP283-58 and pAP281-32may be devoid of the initial Methionine amino-acid, due to processing inthe cytoplasm of E. coli. Cleaved, uncleaved forms of AP205 VLP, ormixtures thereof are further preferred embodiments of the invention.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of a mixture of recombinant coat proteins, orfragments thereof, of the RNA-phage AP205 and of recombinant mutant coatproteins, or fragments thereof, of the RNA-phage AP205.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of fragments of recombinant coat proteins orrecombinant mutant coat proteins of the RNA-phage AP205.

Recombinant AP205 coat protein fragments capable of assembling into aVLP and a capsid, respectively are also useful in the practice of theinvention. These fragments may be generated by deletion, eitherinternally or at the termini of the coat protein and mutant coatprotein, respectively. Insertions in the coat protein and mutant coatprotein sequence or fusions of antigen sequences to the coat protein andmutant coat protein sequence, and compatible with assembly into a VLP,are further embodiments of the invention and lead to chimeric AP205 coatproteins, and particles, respectively. The outcome of insertions,deletions and fusions to the coat protein sequence and whether it iscompatible with assembly into a VLP can be determined by electronmicroscopy.

The particles formed by the AP205 coat protein, coat protein fragmentsand chimeric coat proteins described above, can be isolated in pure formby a combination of fractionation steps by precipitation and ofpurification steps by gel filtration using e.g. Sepharose CL-4B,Sepharose CL-2B, Sepharose CL-6B columns and combinations thereof asdescribed in WO 04/007538, which is incorporated by reference in itsentirety. Other methods of isolating virus-like particles are known inthe art, and may be used to isolate the virus-like particles (VLPs) ofbacteriophage AP205. For example, the use of ultracentrifugation toisolate VLPs of the yeast retrotransposon Ty is described in U.S. Pat.No. 4,918,166, which is incorporated by reference herein in itsentirety.

The crystal structure of several RNA bacteriophages has been determined(Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using suchinformation, one skilled in the art could readily identify surfaceexposed residues and modify bacteriophage coat proteins such that one ormore reactive amino acid residues can be inserted. Thus, one skilled inthe art could readily generate and identify modified forms ofbacteriophage coat proteins which can be used in the practice of theinvention. Thus, variants of proteins which form capsids or capsid-likestructures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17,bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophageMS2) can also be used for the inventive compositions and vaccinecompositions. Further possible examples of modified RNA bacteriophagesas well as variants of proteins and N- and C terminal truncation mutantswhich form capsids or capsid like structures, as well as methods forpreparing such compositions and vaccine compositions, respectively,which are suitable for use in the present invention are described in WO02/056905 on page 50, line 33 to page 52, line 29.

The invention thus includes compositions and vaccine compositionsprepared from proteins which form capsids or VLPs, methods for preparingthese compositions from individual protein subunits and VLPs or capsids,methods for preparing these individual protein subunits, nucleic acidmolecules which encode these subunits, and methods for vaccinatingand/or eliciting immunological responses in individuals using thesecompositions of the present invention.

In another preferred embodiment of the invention, the VLP's are free ofa lipoprotein envelope or a lipoprotein-containing envelope. In afurther preferred embodiment, the VLP's are free of an envelopealtogether.

The lack of a lipoprotein envelope or lipoprotein-containing envelopeand, in particular, the complete lack of an envelope leads to a moredefined virus-like particle in its structure and composition. Such moredefined virus-like particles, therefore, may minimize side-effects.Moreover, the lack of a lipoprotein-containing envelope or, inparticular, the complete lack of an envelope avoids or minimizesincorporation of potentially toxic molecules and pyrogens within thevirus-like particle.

In one embodiment, the invention provides a vaccine composition of theinvention comprising a virus-like particle, wherein preferably saidvirus-like particle is a recombinant virus-like particle. Preferably,the virus-like particle comprises, or alternatively consists essentiallyof, or alternatively consists of, recombinant proteins, or fragmentsthereof, of a RNA-phage, preferably of coat proteins of RNA phages.Alternatively, the recombinant proteins of the virus-like particle ofthe vaccine composition of the invention comprise, or alternativelyconsist essentially of, or alternatively consist of mutant coat proteinsof RNA phages, wherein the RNA-phage is selected from the groupconsisting of: (a) bacteriophage Q(; (b) bacteriophage R17; (c)bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f)bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i)bacteriophage NL95; (k) bacteriophage f2; (1) bacteriophage PP7; and (m)bacteriophage AP205.

In a preferred embodiment, the mutant coat proteins of said RNA phagehave been modified by removal, or by addition of at least one lysineresidue by way of substitution. In another preferred embodiment, themutant coat proteins of said RNA phage have been modified by deletion ofat least one lysine residue or by addition of at least one lysineresidue by way of insertion. In a preferred embodiment, the virus-likeparticle comprises recombinant proteins or fragments thereof, ofRNA-phage Qβ, RNA-phage fr, or RNA-phage AP205.

As previously stated, the invention includes virus-like particles orrecombinant forms thereof. Skilled artisans have the knowledge toproduce such particles and attach antigens thereto. Further preferredembodiments of the present invention hereto are disclosed in the ExampleSection.

In one embodiment, the virus-like particle comprises, or alternativelyconsists essentially of, or alternatively consists of recombinantproteins, or fragments thereof, of the BK virus (BKV), wherein therecombinant proteins comprise, or alternatively consist essentially of,or alternatively consist of proteins having an amino acid sequence ofSEQ ID NO:12. BK virus (BKV) is a non-enveloped double stranded DNAvirus belonging to the polyoma virus subfamily of the papovaviridae. VP1is the major capsid protein of BKV. VP1 has 362 amino acids (SEQ ID NO:12, Gene Bank entry: AAA46882) and is 42 kDa in size. When produced inE. coli, insect cells or yeast VP1 spontaneously forms capsid structures(Salunke D. M., et al., Cell 46(6):895-904 (1986); Sasnauskas, K., etal., Biol. Chem. 380(3):381-6 (1999); Sasnauskas, K., et al., 3rdInternational Workshop “Virus-like particles as vaccines” Berlin,September 26-29 (2001); Touze, A., et al., J Gen Virol. 82(Pt 12):3005-9(2001). The capsid is organized in 72 VPI pentamers forming anicosahedral structure. The capsids have a diameter of approximately 45nm.

In one embodiment, the particles used in compositions of the inventionare composed of a Hepatitis B capsid (core) protein (HBcAg) or afragment of a HBcAg which has been modified to either eliminate orreduce the number of free cysteine residues. Zhou et al. (J. Virol.66:5393 5398 (1992)) demonstrated that HBcAgs which have been modifiedto remove the naturally resident cysteine residues retain the ability toassociate and form multimeric structures. Thus, core particles suitablefor use in compositions of the invention include those comprisingmodified HBcAgs, or fragments thereof, in which one or more of thenaturally resident cysteine residues have been either deleted orsubstituted with another amino acid residue (e.g., a serine residue).

The HBcAg is a protein generated by the processing of a Hepatitis B coreantigen precursor protein. A number of isotypes of the HBcAg have beenidentified and their amino acids sequences are readily available tothose skilled in the art. For example, the HBcAg protein having theamino acid sequence shown in SEQ ID NO: 16 is 185 amino acids in lengthand is generated by the processing of a 212 amino acid Hepatitis B coreantigen precursor protein. This processing results in the removal of 29amino acids from the N terminus of the Hepatitis B core antigenprecursor protein. Similarly, the HBcAg protein that is 185 amino acidsin length is generated by the processing of a 214 amino acid Hepatitis Bcore antigen precursor protein.

In preferred embodiments, vaccine compositions of the invention will beprepared using the processed form of a HBcAg (i.e., a HBcAg from whichthe N terminal leader sequence of the Hepatitis B core antigen precursorprotein have been removed).

Further, when HBcAgs are produced under conditions where processing willnot occur, the HBcAgs will generally be expressed in “processed” form.For example, bacterial systems, such as E. coli, generally do not removethe leader sequences, also referred to as “signal peptides,” of proteinswhich are normally expressed in eukaryotic cells. Thus, when an E. coliexpression system directing expression of the protein to the cytoplasmis used to produce HBcAgs of the invention, these proteins willgenerally be expressed such that the N terminal leader sequence of theHepatitis B core antigen precursor protein is not present.

The preparation of Hepatitis B virus-like particles, which can be usedfor the present invention, is disclosed, for example, in WO 00/32227,and hereby in particular in Examples 17 to 19 and 21 to 24, as well asin WO 01/85208, and hereby in particular in Examples 17 to 19, 21 to 24,31 and 41, and in WO 02/056905. For the latter application, it is inparticular referred to Example 23, 24, 31 and 51. All three documentsare explicitly incorporated herein by reference.

The present invention also includes HBcAg variants which have beenmodified to delete or substitute one or more additional cysteineresidues. Thus, the vaccine compositions of the invention includecompositions comprising HBcAgs in which cysteine residues not present inthe amino acid sequence shown in SEQ ID NO: 16 have been deleted.

It is well known in the art that free cysteine residues can be involvedin a number of chemical side reactions. These side reactions includedisulfide exchanges, reaction with chemical substances or metabolitesthat are, for example, injected or formed in a combination therapy withother substances, or direct oxidation and reaction with nucleotides uponexposure to UV light. Toxic adducts could thus be generated, especiallyconsidering the fact that HBcAgs have a strong tendency to bind nucleicacids. The toxic adducts would thus be distributed between amultiplicity of species, which individually may each be present at lowconcentration, but reach toxic levels when together.

In view of the above, one advantage to the use of HBcAgs in vaccinecompositions which have been modified to remove naturally residentcysteine residues is that sites to which toxic species can bind whenantigens or antigenic determinants are attached would be reduced innumber or eliminated altogether.

A number of naturally occurring HBcAg variants suitable for use in thepractice of the present invention have been identified. Yuan et al., (J.Virol. 73:10122 10128 (1999)), for example, describe variants in whichthe isoleucine residue at position corresponding to position 97 in SEQID NO:25 is replaced with either a leucine residue or a phenylalanineresidue. The amino acid sequences of a number of HBcAg variants, as wellas several Hepatitis B core antigen precursor variants, are disclosed inGenBank reports AAF121240, AF121239, X85297, X02496, X85305, X85303,AF151735, X85259, X85286, X85260, X85317, X85298, AF043593, M20706,X85295, X80925, X85284, X85275, X72702, X85291, X65258, X85302, M32138,X85293, X85315, U95551, X85256, X85316, X85296, AB033559, X59795,X85299, X85307, X65257, X85311, X85301 (SEQ ID NO:26), X85314, X85287,X85272, X85319, AB010289, X85285, AB010289, AF121242, M90520 (SEQ IDNO:27), P03153, AF110999, and M95589, the disclosures of each of whichare incorporated herein by reference. The sequences of the hereinabovementioned Hepatitis B core antigen precursor variants are furtherdisclosed in WO 01/85208 in SEQ ID NOs: 89-138 of the application WO01/85208. These HBcAg variants differ in amino acid sequence at a numberof positions, including amino acid residues which corresponds to theamino acid residues located at positions 12, 13, 21, 22, 24, 29, 32, 33,35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80,81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126,130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ IDNO:28. Further HBcAg variants suitable for use in the compositions ofthe invention, and which may be further modified according to thedisclosure of this specification are described in WO 01/98333, WO00/177158 and WO 00/214478.

HBcAgs suitable for use in the present invention can be derived from anyorganism so long as they are able to enclose or to be coupled orotherwise attached to, in particular as long as they are capable ofpackaging, an unmethylated CpG-containing oligonucleotide and induce animmune response.

As noted above, generally processed HBcAgs (i.e., those which lackleader sequences) will be used in the vaccine compositions of theinvention. The present invention includes vaccine compositions, as wellas methods for using these compositions, which employ the abovedescribed variant HBcAgs.

Further included within the scope of the invention are additional HBcAgvariants which are capable of associating to form dimeric or multimericstructures. Thus, the invention further includes vaccine compositionscomprising HBcAg polypeptides comprising, or alternatively consistingof, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or99% identical to any of the wild-type amino acid sequences, and forms ofthese proteins which have been processed, where appropriate, to removethe N terminal leader sequence.

Whether the amino acid sequence of a polypeptide has an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical toone of the wild-type amino acid sequences, or a subportion thereof, canbe determined conventionally using known computer programs such theBestfit program. When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference amino acid sequence, the parameters are setsuch that the percentage of identity is calculated over the full lengthof the reference amino acid sequence and that gaps in homology of up to5% of the total number of amino acid residues in the reference sequenceare allowed.

The amino acid sequences of the hereinabove mentioned HBcAg variants andprecursors are relatively similar to each other. Thus, reference to anamino acid residue of a HBcAg variant located at a position whichcorresponds to a particular position in SEQ ID NO:28, refers to theamino acid residue which is present at that position in the amino acidsequence shown in SEQ ID NO:28. The homology between these HBcAgvariants is for the most part high enough among Hepatitis B viruses thatinfect mammals so that one skilled in the art would have littledifficulty reviewing both the amino acid sequence shown in SEQ ID NO:28,and that of a particular HBcAg variant and identifying “corresponding”amino acid residues. Furthermore, the HBcAg amino acid sequence shown inSEQ ID NO:27, which shows the amino acid sequence of a HBcAg derivedfrom a virus which infect woodchucks, has enough homology to the HBcAghaving the amino acid sequence shown in SEQ ID NO:28 that it is readilyapparent that a three amino acid residue insert is present in SEQ IDNO:27 between amino acid residues 155 and 156 of SEQ ID NO:28.

The invention also includes vaccine compositions which comprise HBcAgvariants of Hepatitis B viruses which infect birds, as wells as vaccinecompositions which comprise fragments of these HBcAg variants. As oneskilled in the art would recognize, one, two, three or more of thecysteine residues naturally present in these polypeptides could beeither substituted with another amino acid residue or deleted prior totheir inclusion in vaccine compositions of the invention.

As discussed above, the elimination of free cysteine residues reducesthe number of sites where toxic components can bind to the HBcAg, andalso eliminates sites where cross linking of lysine and cysteineresidues of the same or of neighboring HBcAg molecules can occur.Therefore, in another embodiment of the present invention, one or morecysteine residues of the Hepatitis B virus capsid protein have beeneither deleted or substituted with another amino acid residue.Expression and purification of an HBcAg-Lys variant has been describedin Example 24 of WO 02/056905 and the construction of a HBcAg devoid offree cysteine residues and containing an inserted lysine residue hasbeen described in Example 31 of WO 02/056905.

In other embodiments, compositions and vaccine compositions,respectively, of the invention will contain HBcAgs from which the Cterminal region (e.g., amino acid residues 145 185 or 150 185 of SEQ IDNO: 28) has been removed. Thus, additional modified HBcAgs suitable foruse in the practice of the present invention include C terminaltruncation mutants. Suitable truncation mutants include HBcAgs where 1,5, 10, 15, 20, 25, 30, 34, 35, amino acids have been removed from the Cterminus.

HBcAgs suitable for use in the practice of the present invention alsoinclude N terminal truncation mutants. Suitable truncation mutantsinclude modified HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 aminoacids have been removed from the N terminus.

Further HBcAgs suitable for use in the practice of the present inventioninclude N and C terminal truncation mutants. Suitable truncation mutantsinclude HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 amino acidshave been removed from the N terminus and 1, 5, 10, 15, 20, 25, 30, 34,35 amino acids have been removed from the C terminus.

The invention further includes compositions and vaccine compositions,respectively, comprising HBcAg polypeptides comprising, or alternativelyessentially consisting of, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto the above described truncation mutants.

In certain embodiments of the invention, a lysine residue is introducedinto a HBcAg polypeptide, to mediate the binding of the MelanA peptideanalogue of the invention to the VLP of HBcAg. In preferred embodiments,compositions of the invention are prepared using a HBcAg comprising, oralternatively consisting of, amino acids 1-144, or 1-149, 1-185 of SEQID NO:28, which is modified so that the amino acids corresponding topositions 79 and 80 are replaced with a peptide having the amino acidsequence of Gly-Gly-Lys-Gly-Gly (SEQ ID NO:18) resulting in the HBcAgpolypeptide having the sequence shown in SEQ ID NO:29). Thesecompositions are particularly useful in those embodiments where anantigenic determinant is coupled to a VLP of HBcAg. In further preferredembodiments, the cysteine residues at positions 48 and 107 of SEQ IDNO:28 are mutated to serine. The invention further includes compositionscomprising the corresponding polypeptides having amino acid sequencesshown in any of the hereinabove mentioned Hepatitis B core antigenprecursor variants, which also have above noted amino acid alterations.Further included within the scope of the invention are additional HBcAgvariants which are capable of associating to form a capsid or VLP andhave the above noted amino acid alterations. Thus, the invention furtherincludes compositions and vaccine compositions, respectively, comprisingHBcAg polypeptides which comprise, or alternatively consist of, aminoacid sequences which are at least 80%, 85%, 90%, 95%, 97% or 99%identical to any of the wild-type amino acid sequences, and forms ofthese proteins which have been processed, where appropriate, to removethe N terminal leader sequence and modified with above notedalterations.

Compositions or vaccine compositions of the invention may comprisemixtures of different HBcAgs. Thus, these vaccine compositions may becomposed of HBcAgs which differ in amino acid sequence. For example,vaccine compositions could be prepared comprising a “wild type” HBcAgand a modified HBcAg in which one or more amino acid residues have beenaltered (e.g., deleted, inserted or substituted). Further, preferredvaccine compositions of the invention are those which present highlyordered and repetitive antigen arrays, wherein the antigen is a humanmelanoma MelanA peptide analog.

As previously disclosed, the invention is partly based on the surprisingfinding that immunostimulatory substances, preferably immunostimulatorynucleic acids and even more preferably DNA oligonucleotides oralternatively poly (I:C) can be packaged into VLPs. Unexpectedly, thenucleic acids present in VLPs can be replaced specifically by theimmunostimulatory substances, preferably by the immunostimulatorynucleic acids and even more preferably by the DNA-oligonucleotidescontaining CpG motifs or poly (I:C). As an example, the CpG-VLPs aremore immunogenic and elicit more specific effects than their CpG-freecounterparts and induce enhanced B and T cell responses. The immuneresponse against antigens coupled, fused or attached otherwise to theVLPs is similarly enhanced as the immune response against the VLPitself. In addition, the T cell responses against both the VLPs andantigens are especially directed to the Th1 type. Furthermore, thepackaged nucleic acids and CpGs, respectively, are protected fromdegradation, i.e., they are more stable. Moreover, non-specificactivation of cells from the innate immune system is dramaticallyreduced.

The innate immune system has the capacity to recognize invariantmolecular pattern shared by microbial pathogens. Recent studies haverevealed that this recognition is a crucial step in inducing effectiveimmune responses. The main mechanism by which microbial products augmentimmune responses is to stimulate APC, especially dendritic cells toproduce proinflammatory cytokines and to express high levelscostimulatory molecules for T cells. These activated dendritic cellssubsequently initiate primary T cell responses and dictate the type of Tcell-mediated effector function.

Two classes of nucleic acids, namely 1) bacterial DNA that containsimmunostimulatory sequences, in particular unmethylated CpGdinucleotides within specific flanking bases (referred to as CpG motifs)and 2) double-stranded RNA synthesized by various types of virusesrepresent important members of the microbial components that enhanceimmune responses. Synthetic double stranded (ds) RNA such aspolyinosinic-polycytidylic acid (poly I:C) are capable of inducingdendritic cells to produce proinflammatory cytokines and to express highlevels of costimulatory molecules.

A series of studies by Tokunaga and Yamamoto et al. has shown thatbacterial DNA or synthetic oligodeoxynucleotides induce human PBMC andmouse spleen cells to produce type I interferon (IFN) (reviewed inYamamoto et al., Springer Semin Immunopathol. 22:11-19). Poly (I:C) wasoriginally synthesized as a potent inducer of type I IFN but alsoinduces other cytokines such as IL-12.

Preferred ribonucleic acid encompass polyinosinic-polycytidylic aciddouble-stranded RNA (poly I:C). Ribonucleic acids and modificationsthereof as well as methods for their production have been described byLevy, H. B (Methods Enzymol. 1981, 78:242-251), DeClercq, E (MethodsEnzymol. 1981,78:227-236) and Torrence, P. F. (Methods Enzymol1981;78:326-331) and references therein. Further preferred ribonucleicacids comprise polynucleotides of inosinic acid and cytidiylic acid suchpoly (IC) of which two strands forms double stranded RNA. Ribonucleicacids can be isolated from organisms. Ribonucleic acids also encompassfurther synthetic ribonucleic acids, in particular synthetic poly (I:C)oligonucleotides that have been rendered nuclease resistant bymodification of the phosphodiester backbone, in particular byphosphorothioate modifications. In a further embodiment the ribosebackbone of poly (I:C) is replaced by a deoxyribose. Those skilled inthe art know procedures how to synthesize synthetic oligonucleotides.

In another preferred embodiment of the invention molecules that activetoll-like receptors (TLR) are enclosed. Ten human toll-like receptorsare known uptodate. They are activated by a variety of ligands. TLR2 isactivated by peptidoglycans, lipoproteins, lipopolysacchrides,lipoteichonic acid and Zymosan, and macrophage-activating lipopeptideMALP-2; TLR3 is activated by double-stranded RNA such as poly (I:C);TLR4 is activated by lipopolysaccharide, lipoteichoic acids and taxoland heat-shock proteins such as heat shock protein HSP-60 and Gp96; TLR5is activated by bacterial flagella, especially the flagellin protein;TLR6 is activated by peptidoglycans, TLR7 is activated by imiquimoid andimidazoquinoline compounds, such as R-848, loxoribine and bropirimineand TLR9 is activated by bacterial DNA, in particular CpG DNA. Ligandsfor TLR1, TLR8 and TLR10 are not known so far. However, recent reportsindicate that same receptors can react with different ligands and thatfurther receptors are present. The above list of ligands is notexhaustive and further ligands are within the knowledge of the personskilled in the art.

Preferably, the unmethylated CpG-containing oligonucleotide comprisesthe sequence:

5′X1X2CGX3X4 3′

wherein X1, X2, X3 and X4 are any nucleotide. In addition, theoligonucleotide can comprise about 6 to about 100,000 nucleotides,preferably about 6 to about 2000 nucleotides, more preferably about 20to about 2000 nucleotides, and even more preferably comprises about 20to about 300 nucleotides. In addition, the oligonucleotide can comprisemore than 100 to about 2000 nucleotides, preferably more than 100 toabout 1000 nucleotides, and more preferably more than 100 to about 500nucleotides.

In a preferred embodiment, the CpG-containing oligonucleotide containsone or more phosphorothioate modifications of the phosphate backbone.For example, a CpG-containing oligonucleotide having one or morephosphate backbone modifications or having all of the phosphate backbonemodified and a CpG-containing oligonucleotide wherein one, some or allof the nucleotide phosphate backbone modifications are phosphorothioatemodifications are included within the scope of the present invention.Thus, in a preferred embodiment, at least one of the nucleotide X1, X2,X3, and X4 has a phosphate backbone modification.

In a further very preferred embodiment of the present invention, theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein said unmethylated CpG-containingoligonucleotide has a nucleic acid sequence selected from the groupconsisting of (a) GGGGACGATCGTCGGGGGG ((SEQ ID NO: 2); and typicallyabbreviated herein as G3-6), (b) GGGGGACGATCGTCGGGGGG ((SEQ ID NO: 3);and typically abbreviated herein as G4-6), (c) GGGGGGACGATCGTCGGGGGG((SEQ ID NO: 4); and typically abbreviated herein as G5-6), (d)GGGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 5); and typically abbreviated hereinas G6-6), (e) GGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO: 6); and typicallyabbreviated herein as G7-7), (f) GGGGGGGGGACGATCGTCGGGGGGGG ((SEQ ID NO:7); and typically abbreviated herein as G8-8), (g)GGGGGGGGGGACGATCGTCGGGGGGGGG ((SEQ ID NO: 8); and typically abbreviatedherein as G9-9), (h) GGGGGGCGACGACGATCGTCGTCGGGGGGG ((SEQ ID NO: 9); andtypically abbreviated herein as G6), (i) tccatgacgttcctgaataat ((SEQ IDNO: 34); and typically abbreviated herein as CyCpGpt), (j)TCCATGACGTTCCTGAATAAT ((SEQ ID NO: 35); and typically abbreviated hereinCyCpG), (k) tccatgacgttcctgacgtt ((SEQ ID NO: 36); and typicallyabbreviated herein as B-CpGpt), (l) TCCATGACGTTCCTGACGTT ((SEQ ID NO:37); and typically abbreviated herein as B-CpG), (m) ggggtcaacgttgaggggg((SEQ ID NO: 38); and typically abbreviated herein as NKCpOpt), (n)GGGGTCAACGTTGA GGGGG ((SEQ ID NO: 39); and typically abbreviated hereinas NKCpG), (o) attattcaggaacgtcatgga ((SEQ ID NO: 40); and typicallyabbreviated herein as CyCpG-rev-pt), (p) GGGGGGGGGGGACGATCGTCGGGGGGGGGG((SEQ ID NO: 41); and typically abbreviated herein as g10gacga-PO(G10-PO)), (q) gggggggggggacgatcgtcgggggggggg ((SEQ ID NO: 42); andtypically abbreviated herein g10gacga-PS(G10-PS)), (r)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC GCGCGCGAAATGCATGTCAAAGACAGCAT ((SEQ IDNO: 43); and typically abbreviated herein as (CpG)20OpA), (s)TCCATGACGTTCCTGAATAATCGC GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG ((SEQ IDNO: 44); and typically abbreviated herein as Cy(CpG)20), (t)TCCATGACGTTCCTGAATAATCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGAAATGCATGTCAAA GACAGCAT ((SEQ IDNO: 45); and typically abbreviated herein as Cy(CpG)20-OpA), (u)TCCATGACGTTCCTGAATAATAAATGCATGTCAAAGACAGCAT ((SEQ ID NO: 46); andtypically abbreviated herein as CyOpA), (v)TCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTT CCTGAATAAT ((SEQID NO: 47); and typically abbreviated herein as CyCyCy), (w)TCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTGGATGACGTTGGTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCC ((SEQ ID NO: 48); andtypically abbreviated herein as Cy150-1), and (x) CTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATTCATGACTTCCTGAATAATTCCATGACGTTGGTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAAAATTCCAATCAAGCTTATCGATACCGTCGACC (SEQ ID NO: 49), and typically abbreviatedherein as dsCyCpG-253 (complementary strand not shown). Small letters asshown in the afore mentioned sequences of SEQ ID NO: 34 to SEQ ID NO: 49indicate deoxynucleotides connected via phosphorothioate bonds whilelarge letters indicate deoxynucleotides connected via phosphodiesterbonds.

In again further very preferred embodiment of the present invention, theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein said unmethylated CpG-containingoligonucleotide has a nucleic acid sequence ofGGGGGGGGGGGACGATCGTCGGGGGGGGGG ((SEQ ID NO: 41); and typicallyabbreviated herein as g10gacga-PO or G10-PO).

The CpG-containing oligonucleotide can also be recombinant, genomic,synthetic, cDNA, plasmid-derived and single or double stranded. For usein the instant invention, the nucleic acids can be synthesized de novousing any of a number of procedures well known in the art. For example,the β-cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers,M. H., Tet. Let. 22:1859 (1981); nucleoside H-phosphonate method (Garegget al., Tet. Let. 27:4051-4054 (1986); Froehler et al., Nucl. Acid. Res.14:5399-5407 (1986); Garegg et al., Tet. Let. 27:4055-4058 (1986),Gaffney et al., Tet. Let. 29:2619-2622 (1988)). These chemistries can beperformed by a variety of automated oligonucleotide synthesizersavailable in the market. Alternatively, CpGs can be produced on a largescale in plasmids, (see Sambrook, T., et al., “Molecular Cloning: ALaboratory Manual,” Cold Spring Harbor laboratory Press, New York, 1989)which after being administered to a subject are degraded intooligonucleotides. Oligonucleotides can be prepared from existing nucleicacid sequences (e.g., genomic or cDNA) using known techniques, such asthose employing restriction enzymes, exonucleases or endonucleases.

The immunostimulatory substances, the immunostimulatory nucleic acids aswell as the unmethylated CpG-containing oligonucleotide can be bound tothe VLP by any way known is the art provided the composition enhances animmune response in an animal. For example, the oligonucleotide can bebound either covalently or non-covalently. In addition, the VLP canenclose, fully or partially, the immunostimulatory substances, theimmunostimulatory nucleic acids as well as the unmethylatedCpG-containing oligonucleotide. Preferably, the immunostimulatorynucleic acid as well as the unmethylated CpG-containing oligonucleotidecan be bound to a VLP site such as an oligonucleotide binding site(either naturally or non-naturally occurring), a DNA binding site or aRNA binding site. In another embodiment, the VLP site comprises anarginine-rich repeat or a lysine-rich repeat.

One specific use for the compositions of the invention is to activatedendritic cells for the purpose of enhancing a specific immune responseagainst antigens. The immune response can be enhanced using ex vivo orin vivo techniques. The ex vivo procedure can be used on autologous orheterologous cells, but is preferably used on autologous cells. Inpreferred embodiments, the dendritic cells are isolated from peripheralblood or bone marrow, but can be isolated from any source of dendriticcells. Ex vivo manipulation of dendritic cells for the purposes ofcancer immunotherapy have been described in several references in theart, including Engleman, E. G., Cytotechnology 25:1 (1997); VanSchooten, W., et al., Molecular Medicine Today, June, 255 (1997);Steinman, R. M., Experimental Hematology 24:849 (1996); and Gluckman, J.C., Cytokines, Cellular and Molecular Therapy 3:187 (1997).

The dendritic cells can also be contacted with the inventivecompositions using in vivo methods. In order to accomplish this, theCpGs are administered in combination with the VLP optionally coupled,fused or otherwise attached to an antigen directly to a subject in needof immunotherapy. In some embodiments, it is preferred that theVLPs/CpGs be administered in the local region of the tumor, which can beaccomplished in any way known in the art, e.g., direct injection intothe tumor.

A preferred embodiment of the present invention is to provide acomposition for enhancing an immune response in an animal comprising (a)a virus-like particle; (b) at least one immunostimulatory substance; and(c) at least one antigen or antigenic determinant; wherein said antigenor said antigenic determinant is bound to said virus-like particle andwherein said antigen comprises, alternatively consists essentially of,or alternatively consists of a human melanoma MelanA peptide analogue,and wherein said immunostimulatory substance is bound to said virus-likeparticle, and wherein said immunostimulatory substance is anunmethylated CpG-containing oligonucleotide, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotide is part of apalindromic sequence, wherein said palindromic sequence is GACGATCGTC(SEQ ID NO: 1), and wherein said palindromic sequence is flanked at its3′-terminus and at its 5′-terminus by more than two and less than 11guanosine entities or, more preferably by 8-10 guanosine entities, or,most preferably by 10 guanosine entities.

We found that the inventive immunostimulatory substances, i.e. theunmethylated CpG-containing oligonucleotides, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotides are part of apalindromic sequence, wherein the palindromic sequence is GACGATCGTC(SEQ ID NO: 1), and wherein the palindromic sequence is flanked at its3′-terminus and at its 5′-terminus by more than two and less than 11guanosine entities, more preferably by 8-10 guanosine entities, or mostpreferably by 10 guanosine entities, are, in particular, effective atstimulating immune cells in vitro.

In a preferred embodiment of the present invention, the palindromicsequence comprises, or alternatively consist essentially of, oralternatively consists of or is GACGATCGTC (SEQ ID NO: 1), wherein saidpalindromic sequence is flanked at its 5′-terminus by at least 3 and atmost 10 guanosine entities and wherein said palindromic sequence isflanked at its 3′-terminus by at least 6 and at most 10 guanosineentities. In another embodiment, the palindromic sequence is flanked atits 5′-terminus by at least 3 and at most 10 guanosine entities andwherein said palindromic sequence is flanked at its 3′-terminus by atleast 6 and at most 10 guanosine entities.

In a further very preferred embodiment of the present invention, theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated CpG-containing oligonucleotide has a nucleicacid sequence selected from (a) GGGGACGATCGTCGGGGGG ((SEQ ID NO: 2); andtypically abbreviated herein as G3-6), (b) GGGGGACGATCGTCGGGGGG ((SEQ IDNO: 3); and typically abbreviated herein as G4-6), (c)GGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 4); and typically abbreviated hereinas G5-6), (d) GGGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 5); and typicallyabbreviated herein as G6-6), (e) GGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO:6); and typically abbreviated herein as G7-7), (f)GGGGGGGGGACGATCGTCGGGGGGGG ((SEQ ID NO: 7); and typically abbreviatedherein as G8-8), (g) GGGGGGGGGGACGATCGTCGGGGGGGGG ((SEQ ID NO: 8); andtypically abbreviated herein as G9-9), (h)GGGGGGCGACGACGATCGTCGTCGGGGGGG ((SEQ ID NO: 9); and typicallyabbreviated herein as G6), and (i) GGGGGGGGGGGACGATCGTCGGGGGGGGGG ((SEQID NO: 41); and typically abbreviated herein as G10-PO).

In a further preferred embodiment of the present invention theimmunostimulatory substance is an umnethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said palindromic sequence is GACGATCGTC (SEQ ID NO: 1), andwherein said palindromic sequence is flanked at its 5′-terminus by atleast 4 and at most 10 guanosine entities and wherein said palindromicsequence is flanked at its 3′-terminus by at least 6 and at most 10guanosine entities.

In another preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated CpG-containing oligonucleotide has a nucleicacid sequence selected from (a) GGGGGACGATCGTCGGGGGG ((SEQ ID NO: 3);and typically abbreviated herein as G4-6), (b) GGGGGGACGATCGTCGGGGGG((SEQ ID NO: 4); and typically abbreviated herein as G5-6), (c)GGGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 5); and typically abbreviated hereinas G6-6), (d) GGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO: 6); and typicallyabbreviated herein as G7-7), (e) GGGGGGGGGACGATCGTCGGGGGGGG ((SEQ ID NO:7); and typically abbreviated herein as G8-8), (f)GGGGGGGGGGACGATCGTCGGGGGGGGG ((SEQ ID NO: 8); and typically abbreviatedherein as G9-9); and (g) GGGGGGGGGGGACGATCGTCGGGGGGGGGG ((SEQ ID NO:41); and typically abbreviated herein as G10-PO).

In a further preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said palindromic sequence is GACGATCGTC (SEQ ID NO: 1), andwherein said palindromic sequence is flanked at its 5′-terminus by atleast 5 and at most 8 guanosine entities and wherein said palindromicsequence is flanked at its 3′-terminus by at least 6 and at most 10guanosine entities.

The experimental data show that the ease of packaging of the preferredinventive immunostimulatory substances, i.e. the guanosine flanked,palindromic and unmethylated CpG-containing oligonucleotides, whereinthe palindromic sequence is GACGATCGTC (SEQ ID NO: 1), and wherein thepalindromic sequence is flanked at its 3′-terminus and at its5′-terminus by less than 11 or less than 10 guanosine entities, intoVLP's increases if the palindromic sequences are flanked by fewerguanosine entities. However, decreasing the number of guanosine entitiesflanking the palindromic sequences leads to a decrease of stimulatingblood cells in vitro. Thus, packagability is paid by decreasedbiological activity of the indicated inventive immunostimulatorysubstances. The present preferred embodiments represent, thus, acompromise between packagability and biological activity.

In another preferred embodiment of the present invention theimmunostimulatory substance is an umnethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated CpG-containing oligonucleotide has a nucleicacid sequence selected from (a) GGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 4);and typically abbreviated herein as G5-6), (b) GGGGGGGACGATCGTCGGGGGG((SEQ ID NO: 5); and typically abbreviated herein as G6-6), (c)GGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO: 6); and typically abbreviatedherein as G7-7), (d) GGGGGGGGGACGATCGTCGGGGGGGG ((SEQ ID NO: 7); andtypically abbreviated herein as G8-8); and (e)GGGGGGGGGGGACGATCGTCGGGGGGGGGG ((SEQ ID NO: 41); and typicallyabbreviated herein as G10-PO).

In a preferred embodiment of the present invention the immunostimulatorysubstance is an unmethylated CpG-containing oligonucleotide, wherein theCpG motif of said unmethylated CpG-containing oligonucleotide is part ofa palindromic sequence, wherein said unmethylated has the nucleic acidsequence of SEQ ID NO: 7, i.e. the immunostimulatory substance is G8-8,or of SEQ ID NO: 41, i.e. G10-PO.

In a very preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated has the nucleic acid sequence of SEQ ID NO:41, i.e. the immunostimulatory substance is G10-PO. Thus, in a verypreferred embodiment, the present invention provides a composition forenhancing an immune response in an animal comprising (a) a virus-likeparticle; (b) at least one immunostimulatory substance; and (c) at leastone antigen or antigenic determinant; wherein said antigen is bound tosaid virus-like particle and wherein said antigen comprises,alternatively consists essentially of, or alternatively consists of ahuman melanoma MelanA peptide analogue, and wherein saidimmunostimulatory substance is bound to said virus-like particle, andwherein said immunostimulatory substance is an unmethylatedCpG-containing oligonucleotide, wherein the CpG motif of saidunmethylated CpG-containing oligonucleotide is part of a palindromicsequence, wherein said palindromic sequence is GACGATCGTC (SEQ ID NO:1), and wherein said palindromic sequence is flanked at its 3′-terminusand at its 5′-terminus by 10 guanosine entities.

As mentioned above, the optimal sequence used to package into VLPs is acompromise between packagability and biological activity. Taking thisinto consideration, the G8-8 immunostimulatoy substance is a preferred,and the G10-PO immunostimulatory substance a very preferred embodimentof the present invention since they are biologically highly active whilestill reasonably well packaged.

The inventive composition further comprise a human melanoma MelanApeptide analogue of the invention bound to the virus-like particle.

In a further preferred embodiment of the invention, the at least oneMelanA peptide analogue is fused to the virus-like particle. As outlinedabove, a VLP is typically composed of at least one subunit assemblinginto a VLP. Thus, in again a further preferred embodiment of theinvention, the MelanA peptide analogue is fused to at least one subunitof the virus-like particle or of a protein capable of being incorporatedinto a VLP generating a chimeric VLP-subunit-antigen fusion.

Fusion of the MelanA peptide analogue can be effected by insertion intothe VLP subunit sequence, or by fusion to either the N- or C-terminus ofthe VLP-subunit or protein capable of being incorporated into a VLP.Hereinafter, when referring to fusion proteins of a peptide to a VLPsubunit, the fusion to either ends of the subunit sequence or internalinsertion of the peptide within the subunit sequence are encompassed.

Fusion may also be effected by inserting MelanA peptide analoguesequences into a variant of a VLP subunit where part of the subunitsequence has been deleted, that are further referred to as truncationmutants. Truncation mutants may have N- or C-terminal, or internaldeletions of part of the sequence of the VLP subunit. For example, thespecific VLP HBcAg with, for example, deletion of amino acid residues 79to 81 is a truncation mutant with an internal deletion. Fusion ofantigens or antigenic determinants to either the N- or C-terminus of thetruncation mutants VLP-subunits also lead to embodiments of theinvention. Likewise, fusion of an epitope into the sequence of the VLPsubunit may also be effected by substitution, where for example for thespecific VLP HBcAg, amino acids 79-81 are replaced with a foreignepitope. Thus, fusion, as referred to hereinafter, may be effected byinsertion of the MelanA peptide analogue sequence in the sequence of aVLP subunit, by substitution of part of the sequence of the VLP subunitwith the MelanA peptide analogue, or by a combination of deletion,substitution or insertions.

The chimeric MelanA peptide analogue—VLP subunit will be in generalcapable of self-assembly into a VLP. VLP displaying epitopes fused totheir subunits are also herein referred to as chimeric VLPs. Asindicated, the virus-like particle comprises or alternatively iscomposed of at least one VLP subunit. In a further embodiment of theinvention, the virus-like particle comprises or alternatively iscomposed of a mixture of chimeric VLP subunits and non-chimeric VLPsubunits, i.e. VLP subunits not having an antigen fused thereto, leadingto so called mosaic particles. This may be advantageous to ensureformation of, and assembly to a VLP. In those embodiments, theproportion of chimeric VLP-subunits may be 1, 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 95% or higher.

Flanking amino acid residues may be added to either end of the sequenceof the peptide or epitope to be fused to either end of the sequence ofthe subunit of a VLP, or for internal insertion of such peptidicsequence into the sequence of the subunit of a VLP. Glycine and serineresidues are particularly favored amino acids to be used in the flankingsequences added to the peptide to be fused. Glycine residues conferadditional flexibility, which may diminish the potentially destabilizingeffect of fusing a foreign sequence into the sequence of a VLP subunit.

In a specific embodiment of the invention, the VLP is a Hepatitis B coreantigen VLP. Fusion proteins to either the N-terminus of a HBcAg(Neyrinck, S. et al., Nature Med. 5:1157-1163 (1999)) or insertions inthe so called major immunodominant region (MIR) have been described(Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)), WO01/98333), and are preferred embodiments of the invention. Naturallyoccurring variants of HBcAg with deletions in the MIR have also beendescribed (Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001),which is expressly incorporated by reference in its entirety), andfusions to the N- or C-terminus, as well as insertions at the positionof the MIR corresponding to the site of deletion as compared to a wtHBcAg are further embodiments of the invention. Fusions to theC-terminus have also been described (Pumpens, P. and Grens, E.,Intervirology 44:98-114 (2001)). One skilled in the art will easily findguidance on how to construct fusion proteins using classical molecularbiology techniques (Sambrook, J. et al., eds., Molecular Cloning, ALaboratory Manual, 2nd. edition, Cold Spring Habor Laboratory Press,Cold Spring Harbor, N.Y. (1989), Ho et al., Gene 77:51 (1989)). Vectorsand plasmids encoding HBcAg and HBcAg fusion proteins and useful for theexpression of a HBcAg and HBcAg fusion proteins have been described(Pumpens, P. & Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S.et al., Nature Med. 5:1157-1163 (1999)) and can be used in the practiceof the invention. An important factor for the optimization of theefficiency of self-assembly and of the display of the epitope to beinserted in the MIR of HBcAg is the choice of the insertion site, aswell as the number of amino acids to be deleted from the HBcAg sequencewithin the MIR (Pumpens, P. and Grens, E., Intervirology 44:98-114(2001); EP 421 635; U.S. Pat. No. 6,231,864) upon insertion, or in otherwords, which amino acids form HBcAg are to be substituted with the newepitope. For example, substitution of HBcAg amino acids 76-80, 79-81,79-80, 75-85 or 80-81 with foreign epitopes has been described (Pumpens,P. and Grens, E., Intervirology 44:98-114 (2001); EP 421 635; U.S. Pat.No. 6,231,864). HBcAg contains a long arginine tail (Pumpens, P. andGrens, E., Intervirology 44:98-114 (2001)) which is dispensable forcapsid assembly and capable of binding nucleic acids (Pumpens, P. andGrens, E., Intervirology 44:98-114 (2001)). HBcAg either comprising orlacking this arginine tail are both embodiments of the invention.

In a further preferred embodiment of the invention, the VLP is a VLP ofa RNA phage. The major coat proteins of RNA phages spontaneouslyassemble into VLPs upon expression in bacteria, and in particular in E.coli. Specific examples of bacteriophage coat proteins which can be usedto prepare compositions of the invention include the coat proteins ofRNA bacteriophages such as bacteriophage Qβ (SEQ ID NO: 10; PIRDatabase, Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 11;Accession No. AAA16663 referring to Qβ A1 protein) and bacteriophage fr(SEQ ID NO: 13; PIR Accession No. VCBPFR).

In another preferred embodiment, the at least one MelanA peptideanalogue is fused to a Qβ coat protein. Fusion protein constructswherein epitopes have been fused to the C-terminus of a truncated formof the A1 protein of Qβ, or inserted within the A1 protein have beendescribed (Kozlovska, T. M., et al., Intervirology, 39:9-15 (1996)). TheA1 protein is generated by suppression at the UGA stop codon and has alength of 329 aa, or 328 aa, if the cleavage of the N-terminalmethionine is taken into account. Cleavage of the N-terminal methioninebefore an alanine (the second amino acid encoded by the Qβ CP gene)usually takes place in E. coli, and such is the case for N-termini ofthe Qβ coat proteins. The part of the A1 gene, 3′ of the UGA amber codonencodes the CP extension, which has a length of 195 amino acids.Insertion of the at least one MelanA peptide analogue between position72 and 73 of the CP extension leads to further embodiments of theinvention (Kozlovska, T. M., et al., Intervirology 39:9-15 (1996)).Fusion of an MelanA peptide analogue at the C-terminus of a C-terminallytruncated Qβ A1 protein leads to further preferred embodiments of theinvention. For example, Kozlovska et al., (Intervirology, 39: 9-15(1996)) describe Qβ A1 protein fusions where the epitope is fused at theC-terminus of the Qβ CP extension truncated at position 19.

As described by Kozlovska et al. (Intervirology, 39: 9-15 (1996)),assembly of the particles displaying the fused epitopes may require thepresence of both the A1 protein-MelanA-peptide-analogue fusion and thewt CP to form a mosaic particle. However, embodiments comprisingvirus-like particles, and hereby in particular the VLPs of the RNA phageQβ coat protein, which are exclusively composed of VLP subunits havingat least one MelanA peptide analogue fused thereto, are also within thescope of the present invention.

The production of mosaic particles may be effected in a number of ways.Kozlovska et al., Intervirology, 39:9-15 (1996), describe three methods,which all can be used in the practice of the invention. In the firstapproach, efficient display of the fused epitope on the VLPs is mediatedby the expression of the plasmid encoding the Qβ A1 protein fusionhaving a UGA stop codon between CP and CP extension in a E. coli strainharboring a plasmid encoding a cloned UGA suppressor tRNA which leads totranslation of the UGA codon into Trp (pISM3001 plasmid (Smiley B. K.,et al., Gene 134:33-40 (1993))). In another approach, the CP gene stopcodon is modified into UAA, and a second plasmid expressing the A1protein-antigen fusion is cotransformed. The second plasmid encodes adifferent antibiotic resistance and the origin of replication iscompatible with the first plasmid (Kozlovska, T. M., et al.,Intervirology 39:9-15 (1996)). In a third approach, CP and the A1protein-antigen fusion are encoded in a bicistronic manner, operativelylinked to a promoter such as the Trp promoter, as described in FIG. 1 ofKozlovska et al., Intervirology, 39:9-15 (1996).

In a further embodiment, the MelanA peptide analogue is inserted betweenamino acid 2 and 3 (numbering of the cleaved CP, that is wherein theN-terminal methionine is cleaved) of the fr CP, thus leading to anMelanA peptide analogue—fr CP fusion protein. Vectors and expressionsystems for construction and expression of fr CP fusion proteinsself-assembling to VLP and useful in the practice of the invention havebeen described (Pushko P. et al., Prot. Eng. 6:883-891 (1993)). In aspecific embodiment, the MelanA peptide analogue sequence is insertedinto a deletion variant of the fr CP after amino acid 2, whereinresidues 3 and 4 of the fr CP have been deleted.

Fusion of epitopes in the N-terminal protuberant β-hairpin of the coatprotein of RNA phage MS-2 and subsequent presentation of the fusedepitope on the self-assembled VLP of RNA phage MS-2 has also beendescribed (WO 92/13081), and fusion of the MelanA peptide analogue ofthe invention by insertion or substitution into the coat protein of MS-2RNA phage is also falling under the scope of the invention.

In another embodiment of the invention, the MelanA peptide analogues ofthe invention is fused to a capsid protein of papillomavirus. In a morespecific embodiment, the MelanA peptide analogues of the invention isfused to the major capsid protein L1 of bovine papillomavirus type 1(BPV-1). Vectors and expression systems for construction and expressionof BPV-1 fusion proteins in a baculovirus/insect cells systems have beendescribed (Chackerian, B. et al., Proc. Natl. Acad. Sci.USA 96:2373-2378(1999), WO 00/23955). Substitution of amino acids 130-136 of BPV-1 L1with an MelanA peptide analogues of the invention leads to a BPV-1L1-MelanA-peptide-analogue fusion protein, which is a preferredembodiment of the invention. Cloning in a baculovirus vector andexpression in baculovirus infected Sf9 cells has been described, and canbe used in the practice of the invention (Chackerian, B. et al., Proc.Natl. Acad. Sci.USA 96:2373-2378 (1999), WO 00/23955). Purification ofthe assembled particles displaying the fused MelanA peptide analogues ofthe invention can be performed in a number of ways, such as for examplegel filtration or sucrose gradient ultracentrifugation (Chackerian, B.et al., Proc. Natl. Acad. Sci.USA 96:2373-2378 (1999), WO 00/23955).

In a further embodiment of the invention, the MelanA peptide analoguesof the invention are fused to a Ty protein capable of being incorporatedinto a Ty VLP. In a more specific embodiment, the MelanA peptideanalogues of the invention are fused to the p1 or capsid protein encodedby the TYA gene (Roth, J. F., Yeast 16:785-795 (2000)). The yeastretrotransposons Ty1, 2, 3 and 4 have been isolated from SaccharomycesSerevisiae, while the retrotransposon Tf1 has been isolated fromSchizosaccharomyces Pombae (Boeke, J. D. and Sandmeyer, S. B., “YeastTransposable elements,” in The molecular and Cellular Biology of theYeast Saccharomyces: Genome dynamics, Protein Synthesis, and Energetics,p. 193, Cold Spring Harbor Laboratory Press (1991)). Theretrotransposons Ty1 and 2 are related to the copia class of plant andanimal elements, while Ty3 belongs to the gypsy family ofretrotransposons, which is related to plants and animal retroviruses. Inthe Ty1 retrotransposon, the p1 protein, also referred to as Gag orcapsid protein, has a length of 440 amino acids. P1 is cleaved duringmaturation of the VLP at position 408, leading to the p2 protein, theessential component of the VLP.

Fusion proteins to p1 and vectors for the expression of said fusionproteins in Yeast have been described (Adams, S. E., et al., Nature329:68-70 (1987)). So, for example, a MelanA peptide analogue of theinvention may be fused to p1 by inserting a sequence coding for theMelanA peptide analogue into the BamH1 site of the pMA5620 plasmid. Thecloning of sequences coding for foreign epitopes into the pMA5620 vectorleads to expression of fusion proteins comprising amino acids 1-381 ofp1 of Ty1-15, fused C-terminally to the N-terminus of the foreignepitope. Likewise, N-terminal fusion of a MelanA peptide analogues ofthe invention, or internal insertion into the p1 sequence, orsubstitution of part of the p1 sequence are also meant to fall withinthe scope of the invention. In particular, insertion of MelanA peptideanalogues of the invention into the Ty sequence between amino acids30-31, 67-68, 113-114 and 132-133 of the Ty protein p1 (EP06771 11)leads to preferred embodiments of the invention.

Further VLPs suitable for fusion of MelanA or MelanA peptide analoguesof the invention are, for example, Retrovirus-like-particles(WO9630523), HIV2 Gag (Kang, Y. C., et al, Biol. Chem. 380:353-364(1999)), Cowpea Mosaic Virus (Taylor, K. M. et al., Biol. Chem.380:387-392 (1999)), parvovirus VP2 VLP (Rueda, P. et al., Virology263:89-99 (1999)), HBsAg (U.S. Pat. No. 4,722,840, EP0201416B1).

Examples of chimeric VLPs suitable for the practice of the invention arealso those described in Intervirology 39:1 (1996). Further examples ofVLPs contemplated for use in the invention are: HPV-1, HPV-6, HPV-11,HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, Tobacco MosaicVirus. Virus-like particles of SV-40, Polyomavirus, Adenovirus, HerpesSimplex Virus, Rotavirus and Norwalk virus have also been made, andchimeric VLPs of those VLPs are also within the scope of the presentinvention.

As indicated, embodiments comprising antigens fused to the virus-likeparticle by insertion within the sequence of the virus-like particlebuilding monomer are also within the scope of the present invention. Insome cases, antigens can be inserted in a form of the virus-likeparticle building monomer containing deletions. In these cases, thevirus-like particle building monomer may not be able to form virus-likestructures in the absence of the inserted antigen.

In some instances, recombinant DNA technology can be utilized to fuse aheterologous protein to a VLP protein (Kratz, P. A., et al., Proc. Natl.Acad. Sci. USA 96:1915 (1999)). For example, the present inventionencompasses VLPs recombinantly fused or chemically conjugated (includingboth covalently and non covalently conjugations) to an antigen (orportion thereof, preferably at least 10, 20 or 50 amino acids) of thepresent invention to generate fusion proteins or conjugates. The fusiondoes not necessarily need to be direct, but can occur through linkersequences. More generally, in the case that epitopes, either fused,conjugated or otherwise attached to the virus-like particle, are used asantigens in accordance with the invention, spacer or linker sequencesare typically added at one or both ends of the epitopes. Such linkersequences preferably comprise sequences recognized by the proteasome,proteases of the endosomes or other vesicular compartment of the cell.

One way of coupling is by a peptide bond, in which the conjugate can bea contiguous polypeptide, i.e. a fusion protein. In a fusion proteinaccording to the present invention, different peptides or polypeptidesare linked in frame to each other to form a contiguous polypeptide. Thusa first portion of the fusion protein comprises an antigen or immunogenand a second portion of the fusion protein, either N-terminal orC-terminal to the first portion, comprises a VLP. Alternatively,internal insertion into the VLP, with optional linking sequences on bothends of the antigen, can also be used in accordance with the presentinvention.

When HBcAg is used as the VLP, it is preferred that the antigen islinked to the C-terminal end of the HBcAg particle. The hepatitis B coreantigen (HBcAg) exhibiting a C-terminal fusion of the MHC class Irestricted peptide p33 derived from lymphocytic choriomeningitis virus(LCMV) glycoprotein can be and was typically used as a model antigen(HBcAg-p33). The 185 amino acids long wild type HBc protein assemblesinto highly structured particles composed of 180 subunits assumingicosahedral geometry. The flexibility of the HBcAg and other VLPs inaccepting relatively large insertions of foreign sequences at differentpositions while retaining the capacity to form structured capsids iswell documented in the literature. This makes the HBc VLPs attractivecandidates for the design of non-replicating vaccines.

A flexible linker sequence (e.g. a polyglycine/polyserine-containingsequence such as [Gly4 Ser]2 (Huston et al., Meth. Enzymol 203:46-88(1991)) can be inserted into the fusion protein between the antigen andligand. Also, the fusion protein can be constructed to contain an“epitope tag”, which allows the fusion protein to bind an antibody (e.g.monoclonal antibody) for example for labeling or purification purposes.An example of an epitope tag is a Glu-Glu-Phe tripeptide which isrecognized by the monoclonal antibody YL1/2.

The invention also relates to the chimeric DNA which contains a sequencecoding for the VLP and a sequence coding for the MelanA peptideanalogue. The DNA can be expressed, for example, in insect cellstransformed with Baculoviruses, in yeast or in bacteria. There are norestrictions regarding the expression system, of which a large selectionis available for routine use. Preferably, a system is used which allowsexpression of the proteins in large amounts. In general, bacterialexpression systems are preferred on account of their efficiency. Oneexample of a bacterial expression system suitable for use within thescope of the present invention is the one described by Clarke et al., J.Gen. Virol. 71: 1109-1117 (1990); Borisova et al., J. Virol. 67:3696-3701 (1993); and Studier et al., Methods Enzymol. 185:60-89 (1990).An example of a suitable yeast expression system is the one described byEmr, Methods Enzymol. 185:231-3 (1990); Baculovirus systems, which havepreviously been used for preparing capsid proteins, are also suitable.Constitutive or inducible expression systems can be used. By the choiceand possible modification of available expression systems it is possibleto control the form in which the proteins are obtained.

In a specific embodiment of the invention, the antigen to which anenhanced immune response is desired is coupled, fused or otherwiseattached in frame to the Hepatitis B virus capsid (core) protein(HBcAg). However, it will be clear to all individuals in the art thatother virus-like particles can be utilized in the fusion proteinconstruct of the invention.

In a further preferred embodiment of the present invention, the at leastone MelanA peptide analogue is bound to the virus-like particle by atleast one covalent bond. Preferably, the least one MelanA peptideanalogue is bound to the virus-like particle by at least one covalentbond, said covalent bond being a non-peptide bond leading to an orderedand repetitive array and a MelanA peptide analogue—VLP conjugate,respectively. This MelanA peptide analogue array and conjugate,respectively, has typically and preferably a repetitive and orderedstructure since the at least one MelanA peptide analogue is bound to theVLP in an oriented manner. Preferably, equal and more than 120, morepreferably equal and more than 180, even more preferably more than 270,and again more preferably equal and more than 360 MelanA-peptides of theinvention are bound to the VLP. The formation of a repetitive andordered MelanA peptide analogue—VLP array and conjugate, respectively,is ensured by an oriented and directed as well as defined binding andattachment, respectively, of the at least one MelanA peptide analogue tothe VLP as will become apparent in the following. Furthermore, thetypical inherent highly repetitive and organized structure of the VLPsadvantageously contributes to the display of the MelanA peptide analoguein a highly ordered and repetitive fashion leading to a highly organizedand repetitive MelanA peptide analogue—VLP array and conjugate,respectively.

Therefore, the preferred inventive conjugates and arrays, respectively,differ from prior art conjugates in their highly organized structure,dimensions, and in the repetitiveness of the antigen on the surface ofthe array. The preferred embodiment of this invention, furthermore,allows expression of the particle in an expression host guaranteeingproper folding and assembly of the VLP, to which the antigen, i.e. theat least one MelanA peptide analogue of the invention, is then furthercoupled

The present invention discloses methods of binding or association ofMelanA peptide analogue of the invention to VLPs. As indicated, in oneaspect of the invention, the at least one MelanA peptide analogue of theinvention is bound to the VLP by way of chemical cross-linking,typically and preferably by using a heterobifunctional cross-linker.Several hetero-bifunctional cross-linkers are known to the art. Inpreferred embodiments, the hetero-bifunctional cross-linker contains afunctional group which can react with preferred first attachment sites,i.e. with the side-chain amino group of lysine residues of the VLP or atleast one VLP subunit, and a further functional group which can reactwith a preferred second attachment site, i.e. a cysteine residue fusedto the MelanA peptide analogue of the invention and optionally also madeavailable for reaction by reduction. The first step of the procedure,typically called the derivatization, is the reaction of the VLP with thecross-linker. The product of this reaction is an activated VLP, alsocalled activated carrier. In the second step, unreacted cross-linker isremoved using usual methods such as gel filtration or dialysis. In thethird step, the MelanA peptide analogue of the invention is reacted withthe activated VLP, and this step is typically called the coupling step.Unreacted MelanA peptide analogue may be optionally removed in a fourthstep, for example by dialysis. Several hetero-bifunctional cross-linkersare known to the art. These include the preferred cross-linkers SMPH(Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB,Sulfo-SMCC, SVSB, SIA and other cross-linkers available for example fromthe Pierce Chemical Company (Rockford, Ill., USA), and having onefunctional group reactive towards amino groups and one functional groupreactive towards cysteine residues. The above mentioned cross-linkersall lead to formation of a thioether linkage. Another class ofcross-linkers suitable in the practice of the invention is characterizedby the introduction of a disulfide linkage between the MelanA peptideanalogue and the VLP upon coupling. Preferred cross-linkers belonging tothis class include for example SPDP and Sulfo-LC-SPDP (Pierce). Theextent of derivatization of the VLP with cross-linker can be influencedby varying experimental conditions such as the concentration of each ofthe reaction partners, the excess of one reagent over the other, the pH,the temperature and the ionic strength. The degree of coupling, i.e. theamount of antigens or antigenic determinants per subunits of the VLP canbe adjusted by varying the experimental conditions described above tomatch the requirements of the vaccine.

A particularly favored method of binding of antigens or antigenicdeterminants to the VLP, is the linking of a lysine residue on thesurface of the VLP with a cysteine residue on the MelanA peptideanalogue of the invention. In some embodiments, fusion, coupling,attachment or binding of an amino acid linker containing a cysteineresidue, as a second attachment site or as a part thereof, to the MelanApeptide analogue of the invention for coupling to the VLP may berequired. Such constructs comprising said amino acid linker may also beobtained by simple peptide syntheses known in the art.

Therefore, in a further preferred embodiment of the present invention,the antigen or antigenic determinant further comprises an amino acidlinker, wherein preferably said amino acid linker comprises, oralternatively consists of, a second attachment site.

In general, flexible amino acid linkers are favored. Examples of theamino acid linker are selected from the group consisting of: (a) CGG;(b) N-terminal gamma 1-linker; (c) N-terminal gamma 3-linker; (d) Ighinge regions; (e) N-terminal glycine linkers; (f) (G)kC(G)n with n=0-12and k=0-5; (g) N-terminal glycine-serine linkers; (h)(G)kC(G)m(S)l(GGGGS)n with n=0-3, k=0-5, m=0-10, l=0-2 (SEQ ID NO: 62);(i) GGC; (k) GGC-NH2; (1) C-terminal gamma 1-linker; (m) C-terminalgamma 3-linker; (n) C-terminal glycine linkers; (o) (G)nC(G)k withn=0-12 and k=0-5; (p) C-terminal glycine-serine linkers; (q)(G)m(S)l(GGGGS)n(G)oC(G)k with n=0-3, k=0-5, m=0-10, l=0-2, and o=0-8(SEQ ID NO: 63).

Further examples of amino acid linkers are the hinge region ofImmunoglobulins, glycine serine linkers (GGGGS)n (SEQ ID NO: 64), andglycine linkers (G)n all further containing a cysteine residue as secondattachment site and optionally further glycine residues. Typicallypreferred examples of said amino acid linkers are N-terminal gammal:CGDKTHTSPP (SEQ ID NO: 65); C-terminal gamma 1: DKTHTSPPCG (SEQ ID NO:66); N-terminal gamma 3: CGGPKPSTPPGSSGGAP (SEQ ID NO: 67); C-terminalgamma 3: PKPSTPPGSSGGAPGGCG (SEQ ID NO: 68); N-terminal glycine linker:GCGGGG (SEQ ID NO: 69); C-terminal glycine linker: GGGGCG (SEQ ID NO:70); C-terminal glycine-lysine linker: GGKKGC (SEQ ID NO: 71);N-terminal glycine-lysine linker: CGKKGG (SEQ ID NO: 72).

Other amino acid linkers particularly suitable in the practice of theinvention, when a hydrophobic antigen or antigenic determinant is boundto a VLP, are CGKKGG (SEQ ID NO: 73), or CGDEGG (SEQ ID NO: 74) forN-terminal linkers, or GGKKGC (SEQ ID NO: 75) and GGEDGC (SEQ ID NO:76), for the C-terminal linkers. For the C-terminal linkers, theterminal cysteine is optionally C-terminally amidated.

Further linkers useful for this invention are amino acid sequences thatallow the release of the antigenic peptide, i.e. the human melanomaMelanA peptide analogue, from the VLP. Examples for these linkers aredescribed in Toes R E et al. J Exp Med. 2001 Jul. 2;194(1):1-12.Moreover, the PAProC- a prediction algorithm for proteasomal cleavagesmight be used (Nussbaum A K, et. al. Immunogenetics. 2001Mar;53(2):87-94) for prediction of aforementioned amino acid sequencesthat allow the release of the antigenic peptide, i.e. the human melanomaMelanA peptide analogue, from the VLP.

In preferred embodiments of the present invention, GGCG (SEQ ID NO: 77),GGC or GGC-NH2 (“NH2” stands for amidation) linkers at the C-terminus ofthe peptide or CGG at its N-terminus are preferred as amino acidlinkers. In general, glycine residues will be inserted between bulkyamino acids and the cysteine to be used as second attachment site, toavoid potential steric hindrance of the bulkier amino acid in thecoupling reaction. In the most preferred embodiment of the invention,the amino acid linker GGC-NH2 is fused to the C-terminus of the MelanApeptide analogue of the invention.

The cysteine residue added to the MelanA peptide analogue of theinvention has to be in its reduced state to react with thehetero-bifunctional cross-linker on the activated VLP, that is a freecysteine or a cysteine residue with a free sulfhydryl group has to beavailable. In the instance where the cysteine residue to function asbinding site is in an oxidized form, for example if it is forming adisulfide bridge, reduction of this disulfide bridge with e.g. DTT, TCEPor β-mercaptoethanol is required. Low concentrations of reducing agentare compatible with coupling as described in WO 02/05690, higherconcentrations inhibit the coupling reaction, as a skilled artisan wouldknow, in which case the reductand has to be removed or its concentrationdecreased prior to coupling, e.g. by dialysis, gel filtration or reversephase HPLC.

Binding of the MelanA peptide analogue of the invention to the VLP byusing a hetero-bifunctional cross-linker according to the preferredmethods described above, allows coupling of the MelanA peptide analogueof the invention to the VLP in an oriented fashion. Other methods ofbinding the MelanA peptide analogue of the invention to the VLP includemethods wherein the MelanA peptide analogue of the invention iscross-linked to the VLP using the carbodiimide EDC, and NHS. In furthermethods, the MelanA peptide analogue of the invention is attached to theVLP using a homo-bifunctional cross-linker such as glutaraldehyde, DSG,BM[PEO]4, BS3, (Pierce Chemical Company, Rockford, Ill., USA) or otherknown homo-bifunctional cross-linkers with functional groups reactivetowards amine groups or carboxyl groups of the VLP.

Other methods of binding the VLP to an MelanA peptide analogue of theinvention include methods where the VLP is biotinylated, and the MelanApeptide analogue of the invention expressed as a streptavidin-fusionprotein, or methods wherein both the MelanA peptide analogue of theinvention and the VLP are biotinylated, for example as described in WO00/23955. In this case, the MelanA peptide analogue of the invention maybe first bound to streptavidin or avidin by adjusting the ratio ofMelanA peptide analogue of the invention to streptavidin such that freebinding sites are still available for binding of the VLP, which is addedin the next step. Alternatively, all components may be mixed in a “onepot” reaction. Other ligand-receptor pairs, where a soluble form of thereceptor and of the ligand is available, and are capable of beingcross-linked to the VLP or the MelanA peptide analogue of the invention,may be used as binding agents for binding MelanA peptide analogue of theinvention to the VLP. Alternatively, either the ligand or the receptormay be fused to the MelanA peptide analogue of the invention, and somediate binding to the VLP chemically bound or fused either to thereceptor, or the ligand respectively. Fusion may also be effected byinsertion or substitution.

As already indicated, in a favored embodiment of the present invention,the VLP is the VLP of a RNA phage, and in a more preferred embodiment,the VLP is the VLP of RNA phage Qβ coat protein.

One or several antigen molecules, i.e. one or several antigens orantigenic determinants, can be attached to one subunit of the capsid orVLP of RNA phages coat proteins, preferably through the exposed lysineresidues of the VLP of RNA phages, if sterically allowable. A specificfeature of the VLP of the coat protein of RNA phages and in particularof the Qβ coat protein VLP is thus the possibility to couple severalantigens per subunit. This allows for the generation of a dense antigenarray.

In a preferred embodiment of the invention, the binding and attachment,respectively, of the at least one MelanA peptide analogue of theinvention to the virus-like particle is by way of interaction andassociation, respectively, between at least one first attachment site ofthe virus-like particle and at least one second attachment of the MelanApeptide analogue of the invention.

VLPs or capsids of Qβ coat protein display a defined number of lysineresidues on their surface, with a defined topology with three lysineresidues pointing towards the interior of the capsid and interactingwith the RNA, and four other lysine residues exposed to the exterior ofthe capsid. These defined properties favor the attachment of antigens tothe exterior of the particle, rather than to the interior of theparticle where the lysine residues interact with RNA. VLPs of other RNAphage coat proteins also have a defined number of lysine residues ontheir surface and a defined topology of these lysine residues.

In further preferred embodiments of the present invention, the firstattachment site is a lysine residue and/or the second attachmentcomprises sulfhydryl group or a cysteine residue. In a very preferredembodiment of the present invention, the first attachment site is alysine residue and the second attachment is a cysteine residue.

In very preferred embodiments of the invention, the MelanA peptideanalogue of the invention is bound via a cysteine residue, to lysineresidues of the VLP of RNA phage coat protein, and in particular to theVLP of Qβ coat protein.

Another advantage of the VLPs derived from RNA phages is their highexpression yield in bacteria that allows production of large quantitiesof material at affordable cost.

As indicated, the inventive conjugates and arrays, respectively, differfrom prior art conjugates in their highly organized structure,dimensions, and in the repetitiveness of the antigen on the surface ofthe array. Moreover, the use of the VLPs as carriers allow the formationof robust antigen arrays and conjugates, respectively, with variableantigen density. In particular, the use of VLPs of RNA phages, andhereby in particular the use of the VLP of RNA phage Qβ coat proteinallows to achieve very high epitope density. In particular, a density ofmore than 1.5 epitopes per subunit has been reached by coupling apeptide to the VLP of Qβ coat protein (e.g. the human Aβ 1-6 peptide asdescribed in WO 2004/016282). The preparation of compositions of VLPs ofRNA phage coat proteins with a high epitope density can be effectedusing the teaching of this application. In prefered embodiment of theinvention, when a MelanA peptide analogue of the invention is coupled tothe VLP of Qβ coat protein, an average number of MelanA peptide analogueof the invention per subunit of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6 , 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 2.5, 2.6,2.7, 2.8, 2.9, or higher is preferred.

The second attachment site, as defined herein, may be either naturallyor non-naturally present with the MelanA peptide analogue of theinvention. In the case of the absence of a suitable natural occurringsecond attachment site on the MelanA peptide analogue of the invention,such a, then non-natural second attachment can be engineered to theantigen.

As described above, four lysine residues are exposed on the surface ofthe VLP of Qβ coat protein. Typically these residues are derivatizedupon reaction with a cross-linker molecule. In the instance where notall of the exposed lysine residues can be coupled to an antigen, thelysine residues which have reacted with the cross-linker are left with across-linker molecule attached to the ε-amino group after thederivatization step. This leads to disappearance of one or severalpositive charges, which may be detrimental to the solubility andstability of the VLP. By replacing some of the lysine residues witharginines, as in the disclosed Qβ coat protein mutants described below,we prevent the excessive disappearance of positive charges since thearginine residues do not react with the cross-linker. Moreover,replacement of lysine residues by arginines may lead to more definedantigen arrays, as fewer sites are available for reaction to theantigen.

Accordingly, exposed lysine residues were replaced by arginines in thefollowing Qβ coat protein mutants and mutant Qβ VLPs disclosed in thisapplication: Qβ-240 (Lys13-Arg; SEQ ID NO:20), Qβ-250 (Lys 2-Arg,Lys13-Arg; SEQ ID NO: 22) and Qβ-259 (Lys 2-Arg, Lys16-Arg; SEQ IDNO:24). The constructs were cloned, the proteins expressed, the VLPspurified and used for coupling to MelanA peptide analogues of theinvention. Qβ-251; (SEQ ID NO: 23) was also constructed, and guidance onhow to express, purify and couple the VLP of Qβ-251 coat protein can befound throughout the application.

In a further embodiment, we disclose a Qβ mutant coat protein with oneadditional lysine residue, suitable for obtaining even higher densityarrays of antigens. This mutant Qβ coat protein, Qβ-243 (Asn 10-Lys; SEQID NO: 21), was cloned, the protein expressed, and the capsid or VLPisolated and purified, showing that introduction of the additionallysine residue is compatible with self-assembly of the subunits to acapsid or VLP. Thus, MelanA peptide analogue arrays and conjugates,respectively, may be prepared using VLP of Qβ coat protein mutants. Aparticularly favored method of attachment of antigens to VLPs, and inparticular to VLPs of RNA phage coat proteins is the linking of a lysineresidue present on the surface of the VLP of RNA phage coat proteinswith a cysteine residue added to the antigen. In order for a cysteineresidue to be effective as second attachment site, a sulfhydryl groupmust be available for coupling. Thus, a cysteine residue has to be inits reduced state, that is, a free cysteine or a cysteine residue with afree sulfhydryl group has to be available. In the instant where thecysteine residue to function as second attachment site is in an oxidizedform, for example if it is forming a disulfide bridge, reduction of thisdisulfide bridge with e.g. DTT, TCEP or β-mercaptoethanol is required.The concentration of reductand, and the molar excess of reductand overantigen has to be adjusted for each antigen. A titration range, startingfrom concentrations as low as 10 μM or lower, up to 10 to 20 mM orhigher reductand if required is tested, and coupling of the antigen tothe carrier assessed. Although low concentrations of reductand arecompatible with the coupling reaction as described in WO 02/056905,higher concentrations inhibit the coupling reaction, as a skilledartisan would know, in which case the reductand has to be removed or itsconcentration decreased, e.g. by dialysis, gel filtration or reversephase HPLC . Advantageously, the pH of the dialysis or equilibrationbuffer is lower than 7, preferably 6. The compatibility of the low pHbuffer with antigen activity or stability has to be tested.

Epitope density on the VLP of RNA phage coat proteins can be modulatedby the choice of cross-linker and other reaction conditions. Forexample, the cross-linkers Sulfo-GMBS and SMPH typically allow reachinghigh epitope density. Derivatization is positively influenced by highconcentration of reactands, and manipulation of the reaction conditionscan be used to control the number of antigens coupled to VLPs of RNAphage coat proteins, and in particular to VLPs of Qβ coat protein.

Prior to the design of a non-natural second attachment site the positionat which it should be fused, inserted or generally engineered has to bechosen. The selection of the position of the second attachment site may,by way of example, be based on a crystal structure of the antigen. Sucha crystal structure of the antigen may provide information on theavailability of the C- or N-termini of the molecule (determined forexample from their accessibility to solvent), or on the exposure tosolvent of residues suitable for use as second attachment sites, such ascysteine residues. Exposed disulfide bridges, as is the case for Fabfragments, may also be a source of a second attachment site, since theycan be generally converted to single cysteine residues through mildreduction, with e.g. 2-mercaptoethylamine, TCEP, β-mercaptoethanol orDTT. Mild reduction conditions not affecting the immunogenicity of theantigen will be chosen. In general, in the case where immunization witha self-antigen is aiming at inhibiting the interaction of thisself-antigen with its natural ligands, the second attachment site willbe added such that it allows generation of antibodies against the siteof interaction with the natural ligands. Thus, the location of thesecond attachment site will be selected such that steric hindrance fromthe second attachment site or any amino acid linker containing the sameis avoided. In further embodiments, an antibody response directed at asite distinct from the interaction site of the self-antigen with itsnatural ligand is desired. In such embodiments, the second attachmentsite may be selected such that it prevents generation of antibodiesagainst the interaction site of the self-antigen with its naturalligands.

Other criteria in selecting the position of the second attachment siteinclude the oligomerization state of the antigen, the site ofoligomerization, the presence of a cofactor, and the availability ofexperimental evidence disclosing sites in the antigen structure andsequence where modification of the antigen is compatible with thefunction of the self-antigen, or with the generation of antibodiesrecognizing the self-antigen.

In very preferred embodiments, the MelanA peptide analogue of theinvention comprises an added single second attachment site or a singlereactive attachment site capable of association with the firstattachment sites on the core particle and the VLPs or VLP subunits,respectively. This further ensures a defined and uniform binding andassociation, respectively, of the at least one, but typically more thanone, preferably more than 10, 20, 40, 80, 120, 150, 180, 210, 240, 270,300, 360, 400, 450 MelanA peptide analogue of the invention to the coreparticle and VLP, respectively. The provision of a single secondattachment site or a single reactive attachment site on the antigen,thus, ensures a single and uniform type of binding and association,respectively leading to a very highly ordered and repetitive array. Forexample, if the binding and association, respectively, is effected byway of a lysine—(as the first attachment site) and cysteine—(as a secondattachment site) interaction, it is ensured, in accordance with thispreferred embodiment of the invention, that only one cysteine residueper antigen, independent whether this cysteine residue is naturally ornon-naturally present on the antigen, is capable of binding andassociating, respectively, with the VLP and the first attachment site ofthe core particle, respectively.

In some embodiments, engineering of a second attachment site onto theMelanA peptide analogue of the invention require the fusion of an aminoacid linker containing an amino acid suitable as second attachment siteaccording to the disclosures of this invention. Therefore, in apreferred embodiment of the present invention, an amino acid linker isbound to the MelanA peptide analogue of the invention by way of at leastone covalent bond. Preferably, the amino acid linker comprises, oralternatively consists of, the second attachment site. In a furtherpreferred embodiment, the amino acid linker comprises a sulfhydryl groupor a cysteine residue. In another preferred embodiment, the amino acidlinker is cysteine. Some criteria of selection of the amino acid linkeras well as further preferred embodiments of the amino acid linkeraccording to the invention have already been mentioned above.

In another specific embodiment of the invention, the attachment site isselected to be a lysine or cysteine residue that is fused in frame tothe HBcAg. In a preferred embodiment, the antigen is fused to theC-terminus of HBcAg via a three leucine linker.

When an antigen or antigenic determinant is linked to the VLP through alysine residue, it may be advantageous to either substitute or deleteone or more of the naturally resident lysine residues, as well as otherlysine residues present in HBcAg variants.

In many instances, when the naturally resident lysine residues areeliminated, another lysine will be introduced into the HBcAg as anattachment site for an antigen or antigenic determinant. Methods forinserting such a lysine residue are known in the art. Lysine residuesmay also be added without removing existing lysine residues.

The C terminus of the HBcAg has been shown to direct nuclearlocalization of this protein. (Eckhardt et al., J. Virol. 65:575 582(1991)). Further, this region of the protein is also believed to conferupon the HBcAg the ability to bind nucleic acids.

As indicated, HBcAgs suitable for use in the practice of the presentinvention also include N terminal truncation mutants. Suitabletruncation mutants include modified HBcAgs where 1, 2, 5, 7, 9, 10, 12,14, 15, or 17 amino acids have been removed from the N terminus.However, variants of virus-like particles containing internal deletionswithin the sequence of the subunit composing the virus-like particle arealso suitable in accordance with the present invention, provided theircompatibility with the ordered or particulate structure of thevirus-like particle. For example, internal deletions within the sequenceof the HBcAg are suitable (Preikschat, P., et al., J. Gen. Virol.80:1777-1788 (1999)).

Further HBcAgs suitable for use in the practice of the present inventioninclude N- and C terminal truncation mutants. Suitable truncationmutants include HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 aminoacids have been removed from the N terminus and 1, 5, 10, 15, 20, 25,30, 34, 35, 36, 37, 38, 39 40, 41, 42 or 48 amino acids have beenremoved from the C terminus.

Vaccine compositions of the invention can comprise mixtures of differentHBcAgs. Thus, these vaccine compositions can be composed of HBcAgs whichdiffer in amino acid sequence. For example, vaccine compositions couldbe prepared comprising a “wild type” HBcAg and a modified HBcAg in whichone or more amino acid residues have been altered (e.g., deleted,inserted or substituted). In most applications, however, only one typeof a HBcAg will be used.

In a preferred embodiment, the virus-like particle comprises at leastone first attachment site and the antigen or antigenic determinantcomprises at least one second attachment site. Preferably, the firstattachment site comprises, or preferably consists of, an amino group ora lysine residue. The second attachment site is preferably selected fromthe group consisting of (a) an attachment site not naturally occurringwith said antigen or antigenic determinant; and (b) an attachment sitenaturally occurring with said antigen or antigenic determinant. Evenmore preferably, the second attachment site comprises, or preferablyconsists of, a sulfhydryl group or a cysteine residue. In a preferredembodiment, the binding of the antigen or antigenic determinant to thevirus-like particle is effected through association between the firstattachment site and the second attachment site, wherein preferably theassociation is through at least one non-peptide bond, and whereinpreferably the antigen or antigenic determinant and the virus-likeparticle interact through said association to form an ordered andrepetitive antigen array. In one embodiment, the first attachment siteis a lysine residue and the second attachment site is a cysteineresidue. In another embodiment, the first attachment site is an aminogroup and the second attachment site is a sulfhydryl group.

In a specific embodiment of the invention, the antigen, and herein inparticular, the melanoma MelanA peptide analogue, comprises one or morecytotoxic T cell epitopes, Th cell epitopes, or a combination of the twoepitopes. Thus, in one embodiment, the antigen or antigenic determinantcomprises one, two, or more cytotoxic T cell epitopes. In anotherembodiment, the antigen or antigenic determinant comprises one, two, ormore Th cell epitopes. In yet another embodiment, the antigen orantigenic determinant comprises one, two or more cytotoxic T cellepitopes and one, two or more Th cell epitopes.

The natural MelanA/Mart-1 epitopes, and for example the MelanA/Mart-126-35 epitope bind with low affinity to human HLA-2 only. Thus, in vivopresentation of the natural MelanA epitopes and peptides, respectively,upon vaccination may be a limiting factor. This is particularlyimportant if Melan A epitopes and peptides, respectively, bound, coupledor fused to VLPs are used for vaccination, since under these conditions,MelanA peptides load HLA molecules by cross-presentation. The process ofcross-presentation is, however, not as efficient as classical pathwaysof antigen presentation and the affinity of the MelanA peptide for HLAis even more important. Thus, for VLP-based vaccinations, it is verypreferable to use MelanA peptide analogues that bind with relativelyhigh affinity to HLA. Similarly, it may also be advantageous to useMelanA peptide analogues that are recognized with higher affinity by thenatural T cell repertoire of the host. As a general rule, MelanAepitopes and peptide analogues, respectively, are preferred that containanchor residues at the proper positions allowing for efficient bindingto MHC molecules.

Therefore, a further aspect of the present invention and a verypreferred embodiment of the present invention is to provide acomposition for enhancing an immune response in an animal comprising (a)a virus-like particle; and (b) an immunostimulatory substance, whereinsaid immunostimulatory substance is bound to said virus-like particle,and wherein said composition further comprises at least one antigen orantigenic determinant, wherein said antigen or antigenic determinant isbound to said virus-like particle, and wherein said at least one antigenor antigenic determinant comprises, alternatively consists essentiallyof, or alternatively consists of a human melanoma MelanA peptideanalogue, and wherein said human melanoma MelanA peptide analogue isbound to said virus-like particle.

In a preferred embodiment of the present invention, the Melan A peptideanalogue is capable of allowing an efficient binding to MHC molecules.The use of a MelanA peptide analogue, thus, allows, in particular, theintroduction of such anchor residues leading to an improved binding toMHC molecules. The introduction of such anchor residues leading to animproved binding to MHC molecules is in particular advantageous, if thenatural and normal, respectively, MelanA peptide does not contain suchanchor residues or does not contains only such anchor residues which areinferior to the newly introduced anchor residue(s) replacing the naturaland normal, respectively anchor residue.

The modification of the normal human MelanA peptide leading to theMelanA peptide analogue, and hereby preferably the introduction of theseanchor residues is effected either by (i) induced mutation (e.g.chemical induction, irradiation or other procedures known to a personskilled in the art) and subsequent selection of modified peptides withimproved binding to MHC or (ii) of selection of modified peptides withimproved binding to MHC arising from natural mutations arising at anylevel of protein sythesis, including but not limited to mutationsarising at the DNA, transcriptional, RNA or translational level ofprotein expression or (iii) or by systematic or random amino acidexchanges, deletions, substitutions or insertions by using classicalpeptide synthesis known by the person skilled in the art. Theidentification of such anchor residues is typically and preferablyeffected by using the SYFPEITHI database as described by Rammensee etal. in Immunogenetics 50:213-219 (1999). The SYFPEITHI database allowscalculating the efficiency of HLA binding for any peptide of choice andit is possible to optimize the peptides regarding the efficiency of HLAbinding using this program. Alternatively, identification of preferredpeptide analogues can be achieved by MHC-peptide binding assaysinvolving but not limited to whole cell assays of T cell activation orrecognition or MHC upregualtion in mutant cell lines,MHC-tetramer-peptide binding assays, competitive binding assays withlabelled peptides, surface plasmon resonance assays, all known to theperson skilled in the art.

In a further preferred embodiment of the present invention, the MelanApeptide analogue is characterized by two, more preferably by a singleamino acid substitution with respect to the corresponding normal MelanApeptide.

In another preferred embodiment of the present invention, the MelanApeptide analogue is protected from protease or peptidase mediateddegradation. The use of MelanA peptide analogues that are protected fromprotease or peptidase degradation leads to increased stability of thepeptide in vivo after application of the peptide to a subject or and/orto increased stability of the peptide during storage in the presence ofproteases or peptidases. The consequence of this increased stability ismore efficient and prolonged presentation of the human melanoma MelanApeptide analogue on MHC and thus the enhanced stimulation of a specificT cell response.

Preferably, the human MelanA peptide analogue is protected bysubstitution of selected amino acid residues of the natural human MelanApeptide by non natural amino acid derivatives as exemplified in Blanchetet al, J. Immunol. 167:5852-5861 (2001) and references cited therein.This overcomes the limitation typically imposed by the fact thatchemically modified MelanA peptides and MelanA peptide analogues,respectively, may not be recognized by the T cells equally well ascompared to the natural and normal, respectively, MelanA peptide.

In another preferred embodiment, the antigen comprises, alternativelyconsists essentially of, or alternatively consists of a human melanomaMelanA/MART-1 peptide analogue having an amino acid sequence which isselected without limitation from the group consisting of (a) LAGIGILTV(SEQ ID NO: 84); (b) MAGIGILTV (SEQ ID NO: 85), (c) EAMGIGILTV (SEQ IDNO: 86), (d) ELAGIGILTV (SEQ ID NO: 50), (e) EMAGIGILTV (SEQ ID NO: 87),(f) YAAGIGILTV (SEQ ID NO: 88), (g) FAAGIGILTV (SEQ ID NO: 89), (h)GHGHSYTTAE ELAGIGILTV (SEQ ID NO: 51), (i) SYTTAEELAGIGILTVILGVL (SEQ IDNO: 52), and (j) ELAGIGILTVILGVL (SEQ ID NO: 53). In a very preferredembodiment, the antigen comprises, alternatively consists essentiallyof, or alternatively consists of a human melanoma MelanA/MART-1 peptideanalogue having an amino acid sequence which is selected withoutlimitation from the group consisting of (a) LAGIGILTV (SEQ ID NO: 84);(b) MAGIGILTV (SEQ ID NO: 85), (c) EAMGIGILTV (SEQ ID NO: 86), (d)ELAGIGILTV (SEQ ID NO: 50), (e) EMAGIGILTV (SEQ ID NO: 87), (f)YAAGIGILTV (SEQ ID NO: 88), and (g) FAAGIGILTV (SEQ ID NO: 89). Thesepeptide analogues as well as their synthesies have been described byValmori at al., J. Immunol. 160: 1750-1758 (1998). These peptideanalogues show increased relative recognition and target cell lysis byfive different cytotoxic T cell clones raised against the naturalmelanoma peptides.

In a very preferred embodiment of the present invention the humanmelanoma MelanA/MART-1 peptide analogue comprises, alternativelyconsists essentially of, or alternatively consists of the sequenceELAGIGILTV (SEQ ID NO: 50). As indicated throughout the examples thisvery preferred embodiment induces expansion of functionalMelanA-specific CD8+T cells in HLA-A2 transgenic mice and represents agood compromise between HLA-binding and TCR-recognition (cf. Valmori atal., J. Immunol. 160: 1750-1758 (1998)).

In a further preferred embodiment of the present invention the humanmelanoma MelanA/MART-1 peptide analogue with the second attachment sitehas an amino acid sequence selected without limitation from the groupconsisting of (a) CGHGHSYTTAE EAAGIGILTV (SEQ ID NO: 54); and typicallyabbreviated herein as 16-35, (b) CGHGHSYTTAEELAGIGILTV (SEQ ID NO: 55);and typically abbreviated herein as MelanA 16-35 A/L), (c) CGGEAAGIGILTV(SEQ ID NO: 56); and typically abbreviated herein as MelanA 26-35, (d)CGGELAGIGILTV (SEQ ID NO: 57); and typically abbreviated herein asMelanA 26-35 A/L), (e) CSYTTAEELAGIGILTVILGVL (SEQ ID NO: 58); andtypically abbreviated herein as MelanA 20-40 A/L), (f)CGGELAGIGILTVILGVL (SEQ ID NO: 59); and typically abbreviated herein asMelanA 26-40 A/L), (g) ELAGIGILTVGGC (SEQ ID NO: 60); and typicallyabbreviated herein as MelanA 26-35-C A/L), (h) CSPKSLELAGIGILTV (SEQ IDNO: 92), and typically abbreviated herein as CSPKSL-MelanA 26-35 A/L;and (i) ELAGIGILTVILGVLGGC (SEQ ID NO: 93), and typically abbreviatedherein as MelanA 26-40-C A/L. In a very preferred embodiment of thepresent invention the human melanoma MelanA/MART-1 peptide analogue withthe second attachment site has an amino acid sequence selected from (a)CGHGHSYTTAEELAGIGILTV (SEQ ID NO: 55); and typically abbreviated hereinas MelanA 16-35 A/L), (b) CGGELAGIGILTV (SEQ ID NO: 57); and typicallyabbreviated herein as MelanA 26-35 A/L), (c) CSYTTAEELAGIGILTVILGVL (SEQID NO: 58); and typically abbreviated herein as MelanA 20-40 A/L), (d)CGGELAGIGILTVILGVL (SEQ ID NO: 59); and typically abbreviated herein asMelanA 26-40 A/L), (e) ELAGIGILTVGGC (SEQ ID NO: 60); and typicallyabbreviated herein as MelanA 26-35-C A/L).

In another very preferred embodiment of the present invention the humanmelanoma MelanA/MART-1 peptide analogue with the second attachment hasan amino acid sequence of CGHGHSYTTAEELAGIGILTV (SEQ ID NO: 55) (MelanA16-35 A/L). As indicated in Example 21, the inventive vaccinecomposition comprising this very preferred embodiment, i.e. the QβMelanA 16-35 A/L vaccine, is processed by dendritic cells.

In another embodiment of the present invention, the MelanA peptideanalogue of the invention, being coupled, fused or otherwise attached tothe virus-like particle, is a T cell epitope, either a cytotoxic or a Thcell epitope. In a further preferred embodiment, the antigen is acombination of at least two, being either similar or different,preferably different, epitopes, wherein the at least two epitopes arelinked directly or by way of a linking sequence. These epitopes arepreferably selected from the group consisting of cytotoxic and Th cellepitopes described for melanomas (gp100, tyrosinase, MAGE-family orNY-ESO-1).

Thus, in a further preferred embodiment of the present invention, saidantigen comprises, or alternatively further comprises, a cytotoxic Tcell epitope, a Th cell epitope or a combination of at least two of saidepitopes, wherein said at least two epitopes are bound directly or byway of a linking sequence, and wherein preferably said cytotoxic T cellepitope is a viral or a tumor cytotoxic T cell epitope.

Preferred cytotoxic T cell epitopes are MelanA epitopes: (16-36 A/L)(25-36 A/L); Tyrosinase epitopes: (1-9) and (368-376) (Panelli, M. C.et.al., J. Immunol., 2000, 164, 495-504); Gp100 epitopes: (154-162),(209-217 (T210M)), (280-288) and (280-288 (A288V)) and (457-466)(Nielsen, M. B. et. al., 2000. J Immunol., 164 (4)., 2287-96 andLinette, G. P. et. al. J Immunol, 2000, 164, 3402-3412 and Skipper, J.C., Int. J. Cancer, 1999, 82, 669-677 and Pass, H. A., et. al., Cancer JSci Am., 1998, 4, 316-323); TRP2 epitopes: (180-188) (288-296) (455-463)(Sun, Y. et.al., Int. J Cancer, 2000, 87 (3), 399-404 and Parkhurst, M.R. et.al., Cancer Res., 1998, 58, 4895-8901 and Harada, M., et.al.,Cancer Res., 2001, 61, 1089-1094)); NY-ESO-1 epitope: 157-165 (Chen, J.L., et. al., J. Immunol, 2000, 165, 948-955); and MAGE-A epitope:(248V9) (Graff-Dubois, S., et.al., 2002, J. Immunol, 169, 575-580)

A preferred combination of cytotoxic T cell epitopes is the combinationMelanA (25-36 A/L), Tyrosinase (368-376), Gp100 (209-217 (T210M)) and(457-466), and NY-ESO-1 (157-165).

Preferred Th cell epitopes are Mage-3 epitopes: (281-295), (141-155),and (146-160) (Kobayashi, H., et.al., Cancer Res., 2001, 61, 4773-4778);Tyrosinase epitopes: (188-208), (193-203) (Kobayashi H., et.al.,Immunogenetics,1998, 47, 398-403); GP100 epitope: (44-59); and NY-ESO-1epitopes: (115-132), (121-138), (139-156), (119-143), and (134-148)(Zarour, H. M., et.al., Cancer Res., 2002, 62, 213-218).

Preferred combinations of cytotoxic and Th cell epitopes are MelanAepitopes (1-118 A/L) (SEQ ID NO: 94); NY-ESO-1 epitopes 115-165; or thecombination of MelanA (25-36 A7L), gp100 (209-217 (T210M),Mage-3(146-160), and gp100 (44-59).

It should also be understood that a mosaic virus-like particle, e.g. avirus-like particle composed of subunits attached to different antigensand epitopes, respectively, is within the scope of the presentinvention. Such a composition of the present invention can be, forexample, obtained by transforming E. coli with two compatible plasmidsencoding the subunits composing the virus-like particle fused todifferent antigens and epitopes, respectively. In this instance, themosaic virus-like particle is assembled either directly in the cell orafter cell lysis. Moreover, such an inventive composition can also beobtained by attaching a mixture of different antigens and epitopes,respectively, to the isolated virus-like particle.

The MelanA peptide analogue of the present invention, and in particularthe indicated epitope or epitopes, can be synthesized or recombinantlyexpressed and coupled to the virus-like particle, or fused to thevirus-like particle using recombinant DNA techniques. Exemplaryprocedures describing the attachment of antigens to virus-like particlesare disclosed in WO 00/32227, in WO 01/85208 and in WO 02/056905, thedisclosures of which are herewith incorporated by reference in itsentirety.

The invention also provides a method of producing a composition forenhancing an immune response in an animal comprising a VLP and animmunostimulatory substance, preferably an unmethylated CpG-containingoligonucleotide bound to the VLP which comprises incubating the VLP withthe immunostimulatory substance and oligonucleotide, respectively,adding RNase and purifying said composition. Preferably, the methodfurther comprises the step of binding an antigen or antigenicdeterminant to said virus-like particle, wherein said antigen comprises,alternatively consists essentially of, or alternatively consists of ahuman melanoma MelanA peptide analogue. In a preferred embodiment, theanigen or antigenic determinant is bound to the virus-like particlebefore incubating the virus-like particle with the immunostimulatorysubstance. In another preferred embodiment, the anigen or antigenicdeterminant is bound to the virus-like particle after purifying thecomposition. In an equally preferred embodiment, the method comprisesincubating the VLP with RNase, adding the immunostimulatory substanceand oligonucleotide, respectively, and purifying the composition.Preferably, the method further comprises the step of binding an antigenor antigenic determinant to said virus-like particle, wherein saidantigen comprises, alternatively consists essentially of, oralternatively consists of a human melanoma MelanA peptide analogue. In apreferred embodiment, the anigen or antigenic determinant is bound tothe virus-like particle before incubating the virus-like particle withthe RNase. In another preferred embodiment, the anigen or antigenicdeterminant is bound to the virus-like particle after purifying thecomposition. In one embodiment, the VLP is produced in a bacterialexpression system. In another embodiment, the RNase is RNase A.

The invention further provides a method of producing a composition forenhancing an immune response in an animal comprising a VLP bound to animmunostimulatory substance, preferably to an unmethylatedCpG-containing oligonucleotide which comprises disassembling the VLP,adding the immunostimulatory substance and oligonucleotide,respectively, and reassembling the VLP. The method can further compriseremoving nucleic acids of the disassembled VLP and/or purifying thecomposition after reassembly. Preferably, the method further comprisesthe step of binding an antigen or antigenic determinant to thevirus-like particle, wherein said antigen comprises, alternativelyconsists essentially of, or alternatively consists of a human melanomaMelanA peptide analogue. In a preferred embodiment, the anigen orantigenic determinant is bound to the virus-like particle beforedisassembling the virus-like particle. In another preferred embodiment,the anigen or antigenic determinant is bound to the virus-like particleafter reassembling the virus-like particle, and preferably afterpurifying the composition.

The invention also provides vaccine compositions which can be used forpreventing and/or attenuating diseases or conditions. Vaccinecompositions of the invention comprise, or alternatively consist of, animmunologically effective amount of the inventive immune enhancingcomposition together with a pharmaceutically acceptable diluent, carrieror excipient. The vaccine can also optionally comprise an adjuvant.

Thus, in a preferred embodiment, the invention provides a vaccinecomprising an immunologically effective amount of the inventive immuneresponse enhancing composition together with a pharmaceuticallyacceptable diluent, carrier or excipient, wherein the compositioncomprises, (a) a virus-like particle; (b) at least one immunostimulatorysubstance; and (c) at least one antigen or antigenic determinant;wherein the antigen or antigenic determinant is bound to the virus-likeparticle, and wherein the immunostimulatory substance is bound to thevirus-like particle, and wherein the antigen comprises, alternativelyconsists essentially of, or alternatively consists of a human melanomaMelanA peptide analogue. Preferably, the vaccine further comprises anadjuvant.

The invention further provides vaccination methods for preventing and/orattenuating diseases or conditions in animals. In one embodiment, theinvention provides vaccines for the prevention of cancer in a wide rangeof species, particularly mammalian species such as human, monkey, cow,dog, cat, horse, pig, etc., preferably human. Vaccines can be designedto treat all types of cancer, preferably melanomas.

It is well known that homologous prime-boost vaccination strategies withproteins or viruses are most often unsuccessful. Preexisting antibodies,upon re-encountering the antigen, are thought to interfere with theinduction of a memory response. To our surprise, the RNA-phage derivedVLPs, in particular the VLP derived from Qβ, do very efficiently inducea memory CD8⁺ T cell response in a homologous prime-boost vaccinationscheme. In contrast, as described in Example 26, live vaccinia virusimmunizations are very ineffective for the induction of a primary CD8⁺ Tcell response and homologous boosting with vaccinia does hardly lead toan expansion of memory CD8⁺ T cells.

Therefore, in a further aspect, the invention provides a method ofimmunizing or treating an animal comprising priming a T cell response inthe animal by administering an immunologically effective amount of theinventive vaccine. Preferably, the method further comprises the step ofboosting the immune response in the animal, wherein preferably theboosting is effected by administering an immunologically effectiveamount of a vaccine of the invention or an immunologically effectiveamount of a heterologous vaccine, wherein even more preferably theheterologous vaccine is a DNA vaccine, peptide vaccine, recombinantvirus or a dendritic cell vaccine.

Moreover, in again another aspect, the invention further provides amethod of immunizing or treating an animal comprising the steps ofpriming a T cell response in the animal, and boosting a T cell responsein the animal, wherein the boosting is effected by administering animmunologically effective amount of the vaccine of the invention.Preferably, the priming is effected by administering an immunologicallyeffective amount of a vaccine of the invention or an immunologicallyeffective amount of a heterologous vaccine, wherein even more preferablysaid heterologous vaccine is a DNA vaccine, peptide vaccine, recombinantvirus or a dendritic cell vaccine.

Moreover, in again another aspect, the invention further provides for acomposition comprising a virus-like particle, at least oneimmunostimulatory substance; and at least one antigen or antigenicdeterminant; wherein said antigen or antigenic determinant is bound tosaid virus-like particle, and wherein said immunostimulatory substanceis bound to said virus-like particle, and wherein said antigen comprisesa cytotoxic T cell epitope, a Th cell epitope or a combination of atleast two of said epitopes, wherein said at least two epitopes are bounddirectly or by way of a linking sequence, and wherein preferably saidcytotoxic T cell epitope is a viral or a tumor cytotoxic T cell epitope.

In again a further aspect, the present invention provides a composition,typically and preferably for enhancing an immune response in an animalcomprising: (a) a virus-like particle; (b) an immunostimulatorysubstance; wherein said immunostimulatory substance (b) is bound to saidvirus-like particle (a); and (c) an antigen, wherein said antigen ismixed with said virus-like particle (a), and wherein said antigencomprises, alternatively consists essentially of, or alternativelyconsists of a human melanoma MelanA peptide analogue. As used herein,the term “mixed” refers to the combination of two or more substances,ingredients, or elements that are added together, are not chemicallycombined with each other and are capable of being separated. Methods ofmixing antigens with virus-like particles are described in WO 04/000351,which is incorporated herein by reference in its entirety.

As would be understood by one of ordinary skill in the art, whencompositions of the invention are administered to an animal, they can bein a composition which contains salts, buffers, adjuvants or othersubstances which are desirable for improving the efficacy of thecomposition. Examples of materials suitable for use in preparingpharmaceutical compositions are provided in numerous sources includingREMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co.,(1990)).

Various adjuvants can be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art. Further adjuvants that can beadministered with the compositions of the invention include, but are notlimited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS 21,QS 18, CRL1005, Aluminum salts, MF 59, and Virosomal adjuvanttechnology. The adjuvants can also comprise a mixture of thesesubstances.

Compositions of the invention are said to be “pharmacologicallyacceptable” if their administration can be tolerated by a recipientindividual. Further, the compositions of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

The compositions of the present invention can be administered by variousmethods known in the art. The particular mode selected will depend ofcourse, upon the particular composition selected, the severity of thecondition being treated and the dosage required for therapeuticefficacy. The methods of the invention, generally speaking, can bepracticed using any mode of administration that is medically acceptable,meaning any mode that produces effective levels of the active compoundswithout causing clinically unacceptable adverse effects. Such modes ofadministration include oral, rectal, parenteral, intracistemal,intravaginal, intraperitoneal, topical (as by powders, ointments, dropsor transdermal patch), bucal, or as an oral or nasal spray. The term“parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion. The compositionof the invention can also be injected directly in a lymph node.

Components of compositions for administration include sterile aqueous(e.g., physiological saline) or non-aqueous solutions and suspensions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption.

Combinations can be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

Dosage levels depend on the mode of administration, the nature of thesubject, and the quality of the carrier/adjuvant formulation. Typicalamounts are in the range of about 0.1 μg to about 20 mg per subject.Preferred amounts are at least about 1 μg to about 1 mg, more preferablyat least about 10 to about 400 μg per subject. Multiple administrationto immunize the subject is preferred, and protocols are those standardin the art adapted to the subject in question.

The compositions can conveniently be presented in unit dosage form andcan be prepared by any of the methods well-known in the art of pharmacy.Methods include the step of bringing the compositions of the inventioninto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the compositions of the invention into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration can be presented asdiscrete units, such as capsules, tablets or lozenges, each containing apredetermined amount of the compositions of the invention. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, an elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions of the invention described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art.

Other embodiments of the invention include processes for the productionof the compositions of the invention and methods of medical treatmentfor cancer and allergies using said compositions.

Further aspects and embodiments of the present invention will becomeapparent in the following examples and the appended claims.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

All patents and publications referred to herein are expresslyincorporated by reference in their entirety.

EXAMPLE 1

Generation of p33-HBcAg VLPs.

The DNA sequence of HBcAg containing peptide p33 from LCMV is given inSEQ ID NO: 15. The p33-HBcAg VLPs were generated as follows: Hepatitis Bclone pEco63 containing the complete viral genome of Hepatitis B viruswas purchased from ATCC. The gene encoding HBcAg was introduced into theEcoRI/HindIII restriction sites of expression vector pkk223.3(Pharmacia) under the control of a strong tac promoter. The p33 peptide(KAVYNFATM) (SEQ ID NO: 80) derived from lymphocytic choriomeningitisvirus (LCMV) was fused to the C-terminus of HBcAg (1-185) via a threeleucine-linker by standard PCR methods. A clone of E. coli K802 selectedfor good expression was transfected with the plasmid, and cells weregrown and resuspended in 5 ml lysis buffer (10 mM Na2HPO4, 30 mM NaCl,10 mM EDTA, 0.25% Tween-20, pH 7.0). 200 μl of lysozyme solution (20mg/ml) was added. After sonication, 4 μl Benzonase and 10 mM MgCl2 wasadded and the suspension was incubation for 30 minutes at RT,centrifuged for 15 minutes at 15,000 rpm at 4° C. and the supernatantwas retained.

Next, 20% (w/v) (0.2 g/ml lysate) ammonium sulfate was added to thesupernatant. After incubation for 30 minutes on ice and centrifugationfor 15 minutes at 20,000 rpm at 4° C. the supernatant was discarded andthe pellet resuspended in 2-3 ml PBS. 20 ml of the PBS-solution wasloaded onto a Sephacryl S-400 gel filtration column (Amersham PharmaciaBiotechnology AG), fractions were loaded onto a SDS-Page gel andfractions with purified p33-VLP capsids were pooled. Pooled fractionswere loaded onto a Hydroxyapatite column. Flow through (which containspurified p33-VLP capsids) was collected and loaded onto a reducingSDS-PAGE gel for monomer molecular weight analysis. Electron microscopywas performed according to standard protocols.

Thus, the structure of the p33-VLPs was assessed by electron microscopyand SDS PAGE. Recombinantly produced HBcAg wild-type VLPs (composed ofHBcAg [aa 1-185] monomers) and p33-VLPs were loaded onto a SephacrylS-400 gel filtration column (Amersham Pharmacia Biotechnology AG) forpurification. Pooled fractions were loaded onto a Hydroxyapatite column.Flow through (which contains purified p33-VLPs) was collected and loadedonto a reducing SDS-PAGE gel for monomer molecular weight analysis.

Throughout the description the terms p33-HBcAg VLP, HBcAg-p33 VLP,p33-VLPs and HBc33 are used interchangeably.

EXAMPLE 2

Cloning, Expression and Purification of GA VLP

The cDNA of GA phage coat protein was amplified from GA phage by reversetranscription followed by a PCR amplification step, using the RevertAidFirst strand cDNA synthesis Kit (Fermentas). The cDNA was cut with theenzymes NcoI and HindIII, and cloned in vector pQβ185 previously cutwith the same enzymes, leading to plasmid 355.24, harboring GA cDNA. Thesequence of the inserted cDNA was checked by DNA sequencing.

Plasmid 355.24 was transformed in E. coli JM109. Expression wasperformed essentially as described for Qβ VLP. A single colony wasinoculated in LB medium containing 20 mg/L Ampicillin overnight withoutshaking. This inoculum was transferred the next day into a larger flaskcontaining M9 medium supplemented with 1% casaminoacids, 0.2% glucoseand 20 mg/L Ampicillin, and incubated under shaking for 14-20 h.

GA VLP was isolated essentially as described for Qβ VLP. Cells werelysed, and the cleared lysate was loaded onto a Sepharose CL-4B column(Amersham Pharmacia). The eluate was concentrated by ammonium sulphateprecipitation, and rechromatographed onto a Sepharose CL-6B column(Amersham Pharmacia). The final step was either an ultracentrifugationon sucrose gradient (20-50% w/v), or on CsCl. The isolated VLPs weresubsequently dialysed against 20 mM Tris, 150 mM NaCl, pH 8.0.

EXAMPLE 3

Fluorescein Labeled CpG-Containing Oligonucleotides can be Packaged intoBKV, HBcAg and Qβ-VLPs.

VLPs produced in yeast contain small amounts of RNA which can be easilydigested and so eliminated by incubating the VLPs with RNase A. Thehighly active RNase A enzyme has a molecular weight of about 14 kDa andis small enough to enter the VLPs to eliminate the undesired ribonucleicacids. Recombinantly produced BKV VLPs (SEQ ID NO: 12) were concentratedto 1 mg/ml in PBS buffer pH7.2 and incubated in the absence or presenceof RNase A (200 μg/ml, Roche Diagnostics Ltd, Switzerland) for 3 h at37° C. After RNase A digestion BKV VLPs were supplemented with 75nmol/ml 5′-fluorescein labeled phosphorothioate CpG-FAM oligonucleotide(oligonucleotide from SEQ ID NO: 34) and incubated for 3 h at 37° C.Subsequently BKV VLPs were subjected to DNaseI digestion for 3 h at 37°C. (40 u/ml AMPD1, Sigma, Division of Fluka AG, Switzerland) or loadedwithout DNaseI digestion. The samples were complemented with 6-foldconcentrated DNA-loading buffer (10 mM Tris pH7.5, 10% v/v glycerol,0.4% orange G) and run for 1 h at 65 volts in a 0.8% native tris-acetatepH 7.5 agarose gel. Upon staining with ethidium bromide nucleic acidsare detected, while in the absence of ethidium bromide UV excitationleads to fluorescence of the fluorescein-label in the CpG-FAM.

BKV VLPs (15 μg) was analyzed by a native 0.8% agarose gelelectrophoresis after control incubation or after digestion with RNase Aand subsequent incubation with double stranded (ds) DNA (246 bp) (SEQ IDNO: 17), upon staining with ethidium bromide or Coomassie Blue. Thefollowing samples were loaded on the gel: 1: BKV VLPs untreated; 2: BKVVLPs RNase A treated; 3: BKV VLPs treated with RNase A and incubatedwith dsDNA; lane M: Gene Ruler 1 kb DNA ladder (MBI Fermentas GmbH,Heidelberg, Germany).

BKV VLPs (15 μg) was analyzed by a native 0.8% agarose gelelectrophoresis after control incubation or after digestion with RNase Aand subsequent incubation with CpG-oligonucleotides (with phosphate- orwith phosphorothioate (pt) backbone) upon staining with ethidium bromideor Coomassie Blue. The following samples were loaded on the gel: 1: BKVVLPs stock (PBS/50% glycerol); 2: BKV VLPs untreated (PBS buffer); 3:BKV VLPs RNase A treated; 4: BKV VLPs RNase A treated post- dialysis; 5:BKV VLPs RNase A treated with CpG-oligonucleotides; 6: BKV VLPs RNase Atreated with CpG(pt)-oligomers; 7: BKV VLPs RNase A treated withCpG(pt)-oligomers post-dialysis; lane M: Gene Ruler 1 kb DNA ladder (MBIFermentas GmbH, Heidelberg, Germany).

The RNase A digestion leads to a change in migration of the VLP, visibleon Coomassie stained agarose gel, presumably due to the lack of negativecharges from the RNA. Addition of CpG-oligonucleotide restores themigration of BKV VLPs and results in a fluorescent band with the samemigration as the RNA band present in untreated VLPs. This clearly showsthat CpG-FAM oligonucleotides have been packaged into VLPs.

EXAMPLE 4

Large Double Stranded Oligonucleotides can be Packaged into BKV VLPs.

To introduce double stranded (ds) nucleotide sequences, the RNase Atreated recombinant BKV VLPs (Example 3) were supplemented with 50 μg/ml(ds) DNA fragments (246 bp in length, dsDNA, SEQ ID NO: 17) andincubated for 3 h at 37° C. The samples were complemented with 6-foldconcentrated DNA-loading buffer (10 mM Tris pH8.0, 10% v/v glycerol,0.4% orange G) and run for 1 h at 65 volts in a 0.8% native tris-acetatepH8.0 agarose gel. BKV VLPs (15 μg) were loaded on a native 0.8% agarosegel electrophoresis and analyzed after control incubation or afterdigestion with RNase A and subsequent incubation with (ds) DNA uponstaining with ethidium bromide or Coomassie Blue in order to assess thepresence of RNA/DNA or protein. Packaged DNA molecules are visible inthe presence of ethidium bromide as a band with the same migration asthe VLP band visualized with Coomassie Blue.

Addition of (ds) DNA restores the migration of BKV VLPs and results in aDNA band with the same migration as the Coomassie Blue stained VLPs.This clearly shows that (ds) DNA has been packaged into BKV VLPs.

EXAMPLE 5

CpG-Containing Oligonucleotides can be Packaged into BKV VLPs.

To introduce immunostimulatory CpG-oligonucleotides, the RNase A treatedrecombinant BKV VLPs (Example 3) were supplemented with 150 nmol/mlCpG-oligonucleotides CyCpG with phosphodiester backbone or CyCpOpt withphosphorothioate backbone and incubated for 3 h at 37° C. VLPpreparations for mouse immunization were extensively dialysed(10,000-fold diluted) for 24 h against PBS pH7.2 with a 300 kDa MWCOdialysis membrane (Spectrum Medical industries Inc., Houston, USA) toeliminate RNase A and the excess of CpG-oligonucleotides. The sampleswere complemented with 6-fold concentrated DNA-loading buffer (10 mMTris pH7.5, 10% v/v glycerol, 0.4% orange G) and run for 1 h at 65 voltsin a 0.8% native tris-acetate pH7.5 agarose gel. BKV VLPs (15 μg) wereloaded on a native 0.8% agarose gel electrophoresis and analyzed aftercontrol incubation or after digestion with RNase A and subsequentincubation with CpG-oligonucleotides (with phosphodiester- or withphosphorothioate backbone) upon staining with ethidium bromide orCoomassie Blue in order to assess the presence of RNA/DNA or protein andthe reduction of unbound CpG-oligonucleotides after dialysis. UnboundCpG-oligonucleotides are visible as a low molecular weight ethidiumbromide stained band. Addition of CpG-oligonucleotides restores themigration of BKV VLPs and results in a DNA band with the same migrationas the Coomassie Blue stained VLPs. This clearly shows thatCpG-oligonucleotides are packaged into BKV VLPs.

EXAMPLE 6

VLPs Containing CpG-Oligonucleotides (with Phosphorothioate Modificationof the Phosphate Backbone) Induce Enhanced Th1 Directed Immune response.

Female BALB/c mice (three mice per group) were subcutaneously injectedwith 10 μg BKV VLPs containing phosphorothioate CpG-oligonucleotideCyCpGpt (SEQ ID NO: 34). As controls mice were subcutaneously injectedwith either 10 μg of RNase treated BKV VLPs alone or BKV VLPs mixed with0.3 nmol or 20 nmol phosphorothioate CpG-oligonucleotides in 200 μl PBSpH7.2 or were left untreated. BKV VLPs were prepared as described inExample 5 and before immunization extensively purified from unboundCpG-oligonucleotide by dialysis. On day 14 after immunization blood wastaken and IgG1 and IgG2a antibody response to BKV VLPs was determined(see Table 1).

TABLE 1 Mouse IgG1 and IgG2a OD50% antibody titers to BKV VLPs on day 14after immunization with BKV VLPs and phosphorothioate (pt)CpG-oligonucleotides. BKV plus 0.3 BKV plus 20 BKV/0.3 OD 50% titer BKVnmol CpG(pt) nmol CpG(pt) nmol CpG(pt) IgG1 1015 823 <40 340 Stdev 470412 0 241 IgG2a 1190 1142 4193 2596 Stdev 406 1219 1137 1232

Immunization with RNase A treated BKV VLPs containing phosphorothioateCpG-oligonucleotides CyCpGpt results in a decreased IgG1 and anincreased anti-BKV VLP IgG2a titer as compared to immunization with thesame amount (0.3 nmol) of CpG-oligonucleotides mixed with BKV VLPs orBKV VLPs alone. Mice immunized with BKV VLPs mixed with 20 nmolphosphorothioate CpG-oligonucleotide CyCpGpt show very low IgG1 and highIgG2a titers. The decrease in IgG1 titer and the increase in IgG2a titeras compared to controls demonstrates a Th1 cell directed immune responseinduced by phosphorothioate CpG-oligonucleotides packaged in BKV VLPs.Table 1 clearly demonstrates the higher potency of BKV VLPs containingCpG-oligonucleotides packaged within the particles as compared to BKVVLPs simply mixed with CpG-oligonucleotides.

EXAMPLE 7

Immunostimulatory nucleic acids can be packaged into HBcAg VLPscomprising fusion proteins with antigens.

HBcAg VLPs, when produced in E. coli by expressing the Hepatitis B coreantigen fusion protein p33-HBcAg (HBc33) (see Example 1) or the fusionprotein to the peptide P1A (HBcP1A), contain RNA which can be digestedand so eliminated by incubating the VLPs with RNase A.

The gene P1A codes for a protein that is expressed by the mastocytomatumor cell line P815. The dominant CTL epitope, termed P1A peptide,binds to MHC class I (Ld) and the complex is recognized by specific CTLclones (Brändle et al., 1998, Eur. J. Immunol. 28: 4010-4019). Fusion ofpeptide P1A-1 (LPYLGWLVF) ((SEQ ID NO: 90) to the C-terminus of HBcAg(aa 185, see Example 1) was performed by PCR using appropriate primersusing standard molecular biology techniques. A three leucine linker wascloned between the HBcAg and the peptide sequence. Expression wasperformed as described in Example 1. The fusion protein of HBcAg withP1A, termed HBcP1A, formed capsids when expressed in E. coli which couldbe purified similar to the procedure described in Example 1.

Enzymatic RNA hydrolysis: Recombinantly produced HBcAg-p33 (HBc33) andHBcAg-P1A (HBcP1A) VLPs at a concentration of 1.0 mg/ml in 1×PBS buffer(KCl 0.2 g/L, KH2PO4 0.2 g/L, NaCl 8 g/L, Na2HPO4 1.15 g/L) pH 7.4, wereincubated in the presence of 300 μg/ml RNase A (Qiagen AG, Switzerland)for 3 h at 37° C. in a thermomixer at 650 rpm.

Packaging of immunostimulatory nucleic acids: After RNA digestion withRNAse A HBcAg-p33 VLPs were supplemented with 130 nmol/mlCpG-oligonucleotides B-CpG, NKCpG, G10-PO (Table 2). Similarly, the150mer single-stranded Cy150-1 and 253mer double stranded dsCyCpG-253,both containing multiple copies of CpG motifs, were added at 130 nmol/mlor 1.2 nmol/ml, respectively, and incubated in a thermomixer for 3 h at37° C. Double stranded CyCpG-253 DNA was produced by cloning a doublestranded multimer of CyCpG into the EcoRV site of pBluescript KS-. Theresulting plasmid, produced in E. coli XL1-blue and isolated using theQiagen Endofree plasmid Giga Kit, was digested with restrictionendonucleases XhoI and XbaI and resulting restriction products wereseparated by agarose electrophoresis. The 253 bp insert was isolated byelectro-elution and ethanol precipitation. Sequence was verified bysequencing of both strands.

TABLE 2 Terminology and sequences of immunostimulatory nucleic acidsused in the Examples. Small letters indicate deoxynucleotides connectedvia phosphorothioate bonds while large letters indicate deoxynucleotidesconnected via phosphodiester bonds SEQ ID Terminology Sequence NOCyCpGpt tccatgacgttcctgaataat 34 CyCpG TCCATGACGTTCCTGAATAAT 35 B-CpGpttccatgacgttcctgacgtt 36 B-CpG TCCATGACGTTCCTGACGTT 37 NKCpGptggggtcaacgttgaggggg 38 NKCpG GGGGTCAACGTTGAGGGGG 39 CyCpG-rev-ptattattcaggaacgtcatgga 40 g10gacga-PO GGGGGGGGGGGACGATCGTCGGGGGGGG 41(G10-PO) g10gacga-PS gggggggggggacgatcgtcgggggggggg 42 (G10-PS)(CpG)20OpA CGCGCGCGCGCGCGCGCGCGCGCGCGCGCG 43CGCGCGCGCGAAATGCATGTCAAAGACAGCAT Cy(CpG)20TCCATGACGTTCCTGAATAATCGCGCGCGCG 44 CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCy(CpG)20-OpA TCCATGACGTTCCTGAATAATCGCGCGCGCG 45CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGAA ATGCATGTCAAAGACAGCAT CyOpATCCATGACGTTCCTGAATAATAAATGCATGT 46 CAAAGACAGCAT CyCyCyTCCATGACGTTCCTGAATAATTCCATGACGT 47 TCCTGAATAATTCCATGACTGGCCTGAATAATCy150-1 TCCATGACGTTCCTGAATAATTCCATGACGT 48TCCTGAATAATTCCATGACTGGCCTGAATAA TTGGATGACGTTGGTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTGAAAT AATTCCATGACGTTCCTGAATAATTCC dsCyCpG-253CTAGAACTAGTGGATCCCCCGGGCTGCAGATT 49 (complementaryCGATTCATGACTTCCTGAATAATTCCATGACG strand notTTGGTGAATAATCCATGACGTTCCTGAATAAT shown) TCCATGACGTTCCTGAATAATTCCAGACGTTCCTGAATAATTCCATGACGTTCCTGAATAATTC CATGACCTCCTGAATAATTCCATGACGTTCCTGAATAATTCCATGACGTTCCTAAAATTCCAAT CAAGCTTATCGATACCGTCGACC

DNAse I treatment: Packaged HBcAg-p33 VLPs were subsequently subjectedto DNaseI digestion (5 U/ml) for 3 h at 37° C. (DNasel, RNase free FlukaAG, Switzerland) and were extensively dialysed (2× against 200-foldvolume) for 24 h against PBS pH 7.4 with a 300 kDa MWCO dialysismembrane (Spectrum Medical industries Inc., Houston, USA) to eliminateRNAse A and the excess of CpG-oligonucleotides.

Benzonase treatment: Since some single stranded oligodeoxynucleotideswere partially resistant to DNaseI treatment, Benzonase treatment wasused to eliminate free oligonucleotides from the preparation. 100-120U/ml Benzonase (Merck KGaA, Darmstadt, Germany) and 5 mM MgCl2 wereadded and incubated for 3 h at 37° C. before dialysis.

Dialysis: VLP preparations packaged with immunostimulatroy nucleic acidsused in mouse immunization experiments were extensively dialysed (2×against 200 fold volume) for 24 h against PBS pH 7.4 with a 300 kDa MWCOdialysis membrane (Spectrum Medical Industries, Houston, US) toeliminate added enzymes and free nucleic acids.

Analytics of packaging: release of packaged immunostimulatory nucleicacids: To 50 μl capsid solution 1 μl of proteinase K (600 U/ml, Roche,Mannheim, Germany), 3 μl 10% SDS-solution and 6 μl 10fold proteinasebuffer (0.5 M NaCl, 50 mM EDTA, 0.1 M Tris pH 7.4) were added andsubsequently incubated overnight at 37° C. VLPs are completed hydrolysedunder these conditions. Proteinase K was inactivated by heating for 20min at 65° C. 1 μl RNAse A (Qiagen, 100 μg/ml, diluted 250 fold) wasadded to 25 μl of capsid. 2-30 μg of capsid were mixed with 1 volume of2× loading buffer (1×TBE, 42% w/v urea, 12% w/v Ficoll, 0.01%Bromphenolblue), heated for 3 min at 95° C. and loaded on a 10% (foroligonucleotides of about 20 nt length) or 15% (for>than 40 mer nucleicacids) TBE/urea polyacrylamid gel (Invitrogen). Alternatively sampleswere loaded on a 1% agarose gel with 6× loading dye (10 mM Tris pH 7.5,50 mM EDTA, 10% v/v glycerol, 0.4% orange G). TBE/urea gels were stainedwith SYBRGold and agarose gels with stained with ethidium bromide.

The oligonucleotides B-CpG, NKCpG and G10-PO were packaged into HBc33.The analysis of B-CpG packaged into HBc33 VLPs was done on a 1% agarosegel stained with ethidium bromide and Coomassie Blue. Loaded on the gelwere 50 μg of the following samples: 1. HBc33 VLP untreated; 2. HBc33VLP treated with RNase A; 3. HBc33 VLP treated with RNase A and packagedwith B-CpG; 4. HBc33 VLP treated with RNase A, packaged with B-CpG andtreated with DNaseI; 5. HBc33 VLP treated with RNase A, packaged withB-CpG, treated with DNaseI and dialysed; 6.1 kb MBI Fermentas DNAladder. The amount of packaged B-CpG extracted from the VLP was analyzedon a 1.5% agarose gel stained with ethidium bromide: Loaded on gel werethe following samples: 1. 0.5 nmol B-CpG control; 2. 0.5 nmol B-CpGcontrol; 3. B-CpG oligo content HBc33 after phenol/chloroformextraction; 4. B-CpG oligo content HBc33 after phenol/chloroformextraction and RNase A treatment; 5. B-CpG oligo content HBc33 afterphenol/chloroform extraction and DNaseI treatment; 6. empty; 7. MBIFermentas 100 bp DNA ladder.

The analysis of NKCpG packaged into HBc33 VLPs was done on a 1% agarosegel stained with ethidiurn bromide and Coomassie Blue. Loaded on the gelwere 15 μg of the following samples: 1. HBc33 VLP untreated; 2. HBc33VLP treated with RNase A; 3. HBc33 VLP treated with RNase A and packagedwith NKCpG; 4. HBc33 VLP treated with RNase A, packaged with NKCpG,treated with DNaseI and dialysed; 5. 1 kb MBI Fermentas DNA ladder. Theanalysis of the amount of packaged NKCpG extracted from the VLP wasanalyzed on a 15% TBE/urea gel stained with SYBR Gold. Loaded on gelwere the following samples: 1. NKCpG oligo content HBc33 afterproteinase K digestion and RNase A treatment; 2. 20 pmol NKCpG control;3. 10 pmol NKCpG control; 4. 40 pmol NKCpG control.

The analysis of g10gacga-PO packaged into HBc33 VLPs was done on a 1%agarose gel stained with ethidium bromide and Coomassie Blue. Loaded onthe gel were 15 μg of the following samples: 1. 1 kb MBI Fermentas DNAladder; 2. HBc33 VLP untreated; 3. HBc33 VLP treated with RNase A; 4.HBc33 VLP treated with RNase A and packaged with g10gacga-PO; 5. HBc33VLP treated with RNase A, packaged with g10gacga-PO, treated withBenzonase and dialysed.

RNA content in the VLPs was strongly reduced after RNaseA treatmentwhile most of the capsid migrated as a slow migrating smear presumablydue to the removal of the negatively charged RNA. After incubation withan excess of oligonucleotides the capsids contained a higher amount ofnucleic acid than the RNAseA treated capsids and therefore migrated atsimilar velocity as the untreated capsids. Additional treatment withDNAse I or Benzonase degraded the free oligonucleotides whileoligonucleotides packaged in the capsids did not degrade, clearlyshowing packaging of oligonucleotides. In some cases packaging ofoligonucleotides was confirmed by proteinase K digestion afterDNAseI/Benzonase treatment and dialysis. The finding thatoligonucleotides released from the capsid with the procedure describedabove were of the same size than the oligonucleotide used for packagingclearly demonstrated packaging of oligonucleotides.

Large single-stranded oligonucleotide Cy150-1 was packaged into HBc33.Cy150-1 contains 7.5 repeats of CyCpG and was synthesized accordingstandard oligonucleotide synthesis methods (IBA, Göttingen, Germany).The analysis of Cy150-1 packaged into HBc33 VLPs was analyzed on a 1%agarose gel stained with ethidium bromide and Coomassie Blue. Loaded onthe gel were 15 μg of the following samples: 1. 1 kb MBI Fermentas DNAladder; 2. HBc33 VLP untreated; 3. HBc33 VLP treated with RNase A; 4.HBc33 VLP treated with RNase A and packaged with Cy150-1; 5. HBc33 VLPtreated with RNase A, packaged with Cy150-1, treated with DNasel anddialysed; 6. HBc33 VLP treated with RNase A, packaged with Cy150-1,treated with DNaseI and dialysed. The analysis of the amount of packagedCy150-1 extracted from the VLP was analyzed on a 10% TBE/urea gelstained with SYBR Gold. Loaded on gel are the following samples: 1. 20pmol Cy150-1 control; 2. 10 pmol Cy150-1 control; 3. 4 pmol Cy150-1control; 4. Cy150-1 oligo content of 4 μg HBc33 after 3 min at 95° C.with 1 volume TBE/urea sample buffer. RNA content in the capsid wasstrongly reduced after RNaseA treatment while most of the capsidmigrated as a slow migrating smear. Capsid were diluted with 4 volumesof water and concentrated to 1 mg/ml. After incubation with an excess ofCy150-1 the capsid contained a bigger amount of nucleic acid and thusmigrated at similar velocity as the untreated capsids. Additionaltreatment with DNAseI degraded the free, not packaged oligonucleotideswhile oligonucleotides in capsids were not degraded. Release of theDNAseI-resistant nucleic acid from the packaged VLPs by heating for 3min at 95° C. in TBE/urea loading buffer revealed the presence of the150 mer.

The oligonucleotide NKCpGpt was also packaged into HBcP1A. The analysisof NKCpGpt packaged into HBcP1A VLPs was done on a 1% agarose gelstained with ethidium bromide and Coomassie Blue. Loaded on the gel were15 μg of the following samples: 1. 1 kb MBI Fermentas DNA ladder; 2.HBcP1A VLP untreated; 3. HBcP1A VLP treated with RNase A; 4. HBcP1A VLPtreated with RNase A and packaged with NKCpGpt. Treatment with RNAsereduced nucleic acid content and slowed migration of the capsids.Addition of NKCpGpt restored nucleic acid content in capsids and fastmigration.

EXAMPLE 8

Immunostimulatory Nucleic Acids can be Packaged in HBcAg-wt Coupled withAntigens.

Recombinantly produced HBcAg-wt VLPs were packaged after coupling withpeptide p33 (CGG-KAVYNFATM) (SEQ ID NO: 81), derived from lymphocyticchoriomeningitis virus (LCMV). For coupling HBcAg-wt VLPs (2 mg/ml) werederivatized with 25× molar excess of SMPH(Succinimidyl-6-[(β-maleimido-propionamido)hexanoate], Pierce) for 1 hat 25° C. in a thermomixer. The derivatized VLPs were dialyzed to Mesbuffer (2-(N-morpholino) ethanesulphonic acid) pH 7.4 for 2×2 h usingMWCO 10.000 kD dialysis membranes at 4° C. VLPs (50 μM) weresubsequently coupled to the N-terminal cysteine of the p33 peptide (250μM) during a 2 h incubation in a thermomixer at 25° C. Samples weredialyzed (MWCO 300.000) extensively to 1× PBS pH 7.4 to eliminateundesired free peptide.

HBcAg-wt VLPs derivatization with SMPH and coupling to p33 peptide wasanalyzed on SDS-PAGE. Samples were analysed by 16% SDS PAGE and stainedwith Coomassie Blue. Loaded on the gel were the following samples: 1.NEBPrestained Protein Marker, Broad Range (# 7708S), 10 μl; 2. p33 peptide;3. HBcAg-wt VLP derivatized with SMPH, before dialysis; 4. HBcAg-wt VLPderivatized with SMPH, after dialysis; 5. HBcAg-wt VLP coupled with p33,supernatant; 6. HBcAg-wt VLP coupled with p33, pellet. HBcAg-wt wasvisible as a 21 kD protein band. Due to the low molecular weight of SMPHis the derivatised product only slightly larger and can not bedistinguished by SDS-PAGE. Peptide alone was visible as a 3 kD band andcoupled product, termed HBx33, showed a strong secondary band atapproximately 24 kD accounting for more than 50% of total HBcAg-wt.

Enzymatic RNA hydrolysis: HBx33 VLPs (0.5-1.0 mg/ml, 1×PBS buffer pH7.4)in the presence of RNase A (300 μg/ml, Qiagen AG, Switzerland) werediluted with 4 volumes H2O to decrease salt concentration to a final0.2×PBS concentration and incubated for 3 h at 37° C. in a thermomixerat 650 rpm.

Packaging of immunostimulatory nucleic acids: After RNase A digestionHBx33 VLPs were concentrated using Millipore Microcon or Centriplusconcentrators, then supplemented with 130 nmol/ml CpG-oligonucleotideB-CpGpt and incubated in a thermomixer for 3 h at 37° C. in 0.2×PBS pH7.4. Subsequently, reaction mixtures were subjected to DNaseI digestion(5 U/ml) for 3 h at 37° C. (DNaseI, RNase free Fluka AG, Switzerland).VLP preparations for mouse immunization were extensively dialysed (2×against 200-fold volume) for 24 h against PBS pH 7.4 with a 300 kDa MWCOdialysis membrane (Spectrum Medical industries Inc., Houston, USA) toeliminate RNase A and the excess of CpG-oligonucleotides. The analysisof B-CpGpt packaged into HBx33 VLPs was done on a 1% agarose gel stainedwith ethidium bromide and Coomassie Blue. Loaded on the gel were 50 μgof the following samples: 1. HBx33 VLP untreated; 2. HBx33 VLP treatedwith RNase A; 3. HBx33 VLP treated with RNase A and packaged withB-CpGpt; 4. HBx33 VLP treated with RNase A, packaged with B-CpGpt andtreated with DNaseI; 5. HBx33 VLP treated with RNase A, packaged withB-CpGpt, treated with DNaseI and dialysed; 6. 1 kb MBI Fermentas DNAladder. It could be shown that RNAse treatment reduced the nucleic acidcontent of the capsids and slowed their migration. Addition of B-CpGptrestored nucleic acid content and fast migration of capsids. DNAse Ionly digested the free oligonucleotides while the packagedoligonucleotides remained in the VLP also after dialysis.

EXAMPLE 9

Immunostimulatory Nucleic Acids can be Packaged into Qβ VLPs Coupledwith Antigens.

Coupling of p33 peptides to Qβ VLPs:

Recombinantly produced virus-like particles of the RNA-bacteriophage Qb(Qβ VLPs) were used untreated or after coupling to p33 peptidescontaining an N-terminal CGG or and C-terminal GGC extension(CGG-KAVYNFATM (SEQ ID NO: 81) and KAVYNFATM-GGC (SEQ ID NO: 82)).Recombinantly produced Qβ VLPs were derivatized with a 10 molar excessof SMPH (Pierce) for 0.5 h at 25° C., followed by dialysis against 20 mMHEPES, 150 mM NaCl, pH 7.2 at 4° C. to remove unreacted SMPH. Peptideswere added in a 5 fold molar excess and allowed to react for 2 h in athermomixer at 25° C. in the presence of 30% acetonitrile. The analysisof the p33 coupling to Qb VLPs was done on SDS-PAGE after Coomassie Bluestaining. Loaded were the following samples: (A) 1. NEB PrestainedProtein Marker, Broad Range (# 7708S), 10 μl; 2. Qb VLP, 14 μg; 3. QbVLP derivatized with SMPH, after dialysis; 4. Qb VLP coupled withCGG-p33, supernatant. (B) 1. NEB Prestained Protein Marker, Broad Range(# 7708S), 10 μl; 2. Qb VLP, 10 μg; 3. Qb VLP coupled with GGC-p33,supernatant. The SDS-PAGE analysis demonstrated multiple coupling bandsconsisting of one, two or three peptides coupled to the Qβ monomer. Forthe sake of simplicity the coupling product of the peptide p33 and QβVLPs was termed, in particular, throughout the example section Qbx33.

Qβ VLPs, when produced in E. coli by expressing the bacteriophage Qβcapsid protein, contain RNA which can be digested and so eliminated byincubating the VLPs with RNase A.

Low Ionic Strength and Low Qβ Concentration Allow RNA Hydrolysis of QβVLPs by RNAse A:

Qβ VLPs at a concentration of 1.0 mg/ml in 20 mM Hepes/150 mM NaClbuffer (HBS) pH 7.4 were either digested directly by addition of RNase A(300 μg/ml, Qiagen AG, Switzerland) or were diluted with 4 volumes H2Oto a final 0.2×HBS concentration and then incubated with RNase A (60μg/ml, Qiagen AG, Switzerland). Incubation was allowed for 3 h at 37° C.in a thermomixer at 650 rpm. RNA hydrolysis from Qb VLPs by RNase Aunder low and high ionic strength was analyzed on a 1% agarose gelstained with ethidium bromide and Coomassie Blue. Loaded on the gel werethe following samples: (A, B) 1. MBI Fermentas 1 kb DNA ladder; 2. QbVLP untreated; 3. Qb VLP treated with RNase A in 1×HBS buffer pH7.2. (C,D) 1. MBI Fermentas 1 kb DNA ladder; 2. Qb VLP untreated; 3. Qb VLPtreated with RNase A in 0.2× HBS buffer pH7.2. It was demonstrated thatin 1× HBS only a very weak reduction of RNA content was observed, whilein 0.2× HBS most of the RNA were hydrolysed. In agreement, capsidmigration was unchanged after addition of RNAse A in 1× HBS, whilemigration was slower after addition of RNAse in 0.2× HBS.

Low Ionic Strength Increases Nucleic Acid Packaging in Qβ VLPs:

After RNase A digestion in 0.2× HBS the Qβ VLPs were concentrated to 1mg/ml using Millipore Microcon or Centriplus concentrators and aliquotswere dialysed against 1× HBS or 0.2× HBS. Qβ VLPs were supplemented with130 nmol/ml CpG-oligonucleotide B-CpG and incubated in a thermomixer for3 h at 37° C. Subsequently Qβ VLPs were subjected to Benzonase digestion(100 U/ml) for 3 h at 37° C. Samples were analysed on 1% agarose gelsafter staining with ethidium bromide or Coomassie Blue. Loaded on thegel were the following samples: 1. Qb VLP untreated; 2. Qb VLP treatedwith RNase A; 3. Qb VLP treated with RNase A and packaged with B-CpG in0.2× HBS buffer pH7.2 and treated with Benzonase; 4. HBx33 VLP (seeexample 12) treated with RNase A, packaged with B-CpG in 1× HBS bufferpH7.2 and treated with Benzonase. In 1× HBS only a very low amount ofoligonucleotides could be packaged, while in 0.2×HBS a strong ethidiumbromide stained band was detectable, which colocalized with theCoomassie blue stain of the capsids.

Different Immunostimulatory Nucleic Acids can be Packaged in Qβ andQbx33 VLPs:

After RNase A digestion in 0.2× HBS the Qβ VLPs or Qbx33 VLPs wereconcentrated to 1 mg/ml using Millipore Microcon or Centriplusconcentrators and supplemented with 130 nmol/ml CpG-oligonucleotidesB-CpGpt, g10gacga and the 253 mer dsCyCpG-253 (Table 2) and incubated ina thermomixer for 3 h at 37° C. Subsequently Qβ VLPs or Qbx33 VLPs weresubjected to DNAse I digestion (5 U/ml) or Benzonase digestion (100U/ml) for 3 h at 37° C. Samples were analysed on 1% agarose gels afterstaining with ethidium bromide or Coomassie Blue.

Loaded on the gel were 50 μg of the following samples: 1. Qbx33 VLPuntreated; 2. Qbx33 VLP treated with RNase A; 3. Qbx33 VLP treated withRNase A and packaged with B-CpGpt; 4. Qbx33 VLP treated with RNase A,packaged with B-CpGpt, treated with DNaseI and dialysed; 5. 1 kb MBIFermentas DNA ladder. (C) depicts the analysis of the amount of packagedoligo extracted from the VLP on a 15% TBE/urea stained with SYBR Gold.Loaded on gel are the following samples: 1. BCpGpt oligo content of 2 μgQbx33 VLP after proteinase K digestion and RNase A treatment; 2. 20 pmolB-CpGpt control; 3. 10 pmol B-CpGpt control; 4. 5 pmol B-CpGpt control.

Loaded on another gel were 15 μg of the following samples: 1. MBIFermentas 1 kb DNA ladder; 2. Qbx33 VLP untreated; 3. Qbx33 VLP treatedwith RNase A; 4. Qbx33 VLP treated with RNase A and packaged withg10gacga-PO; 5. Qbx33 VLP treated with RNase A, packaged withg10gacga-PO, treated with Benzonase and dialysed.

Loaded on a third gel were 15 μg of the following samples: 1. MBIFermentas 1 kb DNA ladder; 2. Qbx33 VLP untreated; 3. Qbx33 VLP treatedwith RNase A; 4. Qbx33 VLP treated with RNase A, packaged withdsCyCpG-253 and treated with DNaseI; 5. Qbx33 VLP treated with RNase A,packaged with dsCyCpG-253, treated with DNaseI and dialysed.

The different nucleic acids B-CpGpt, g10gacga and the 253mer dsDNA couldbe packaged into Qbx33. Packaged nucleic acids were resistant to DNAse Idigestion and remained packaged during dialysis. Packaging of B-CpGptwas confirmed by release of the nucleic acid by proteinase K digestionfollowed by agarose electrophoresis and ethidium bromide staining.

EXAMPLE 10

AP205 Disassembly-Purification-Reassembly and Packaging ofImmunostimulatory Nucleic Acids.

-   -   A. Disassembly and Reassembly of AP205 VLP from Material able to        Reassemble without Addition of Oligonucleotide

Disassembly: 40 mg of lyophilized purified AP205 VLP (SEQ-ID: 80 or 81)were resolubilized in 4 ml 6 M GuHCl, and incubated overnight at 4° C.The disassembly mixture was centrifuged at 8000 rpm (Eppendorf 5810 R,in fixed angle rotor F34-6-38, used in all the following steps). Thepellet was resolubilized in 7 M urea, while the supernatant was dialyzed3 days against NET buffer (20 mM Tris-HCI, pH 7.8 with 5 mM EDTA and 150mM NaCl) with 3 changes of buffer. Alternatively, dialysis was conductedin continuous mode over 4 days. The dialyzed solution was centrifuged at8000 rpm for 20 minutes, and the pellet was resolubilized in 7 M urea,while the supernatant was pelletted with ammonium sulphate (60%saturation), and resolubilized in a 7 M urea buffer containing 10 mMDTT. The previous pellets all resolubilized in 7 M urea were joined, andprecipitated with ammonium sulphate (60% saturation), and resolubilizedin a 7 M urea buffer containing 10 mM DTT. The materials resolubilizedin the 7 M urea buffer containing 10 mM DTT were joined and loaded on aSephadex G75 column equilibrated and eluted with the 7 M urea buffercontaining 10 mM DTT at 2 ml/h. One peak eluted from the column.Fractions of 3 ml were collected. The peak fractions containing AP205coat protein were pooled and precipitated with ammonium sulphate (60%saturation). The pellet was isolated by centrifugation at 8000 rpm, for20 minutes. It was resolubilized in 7 M urea, 10 mM DTT, and loaded on ashort Sepharose 4B column (1.5×27 cm Sepharose 4B, 2 ml/h, 7 M urea, 10mM DTT as elution buffer). Mainly one peak, with a small shoulder elutedfrom the column. The fractions containing the AP205 coat protein wereidentified by SDS-PAGE, and pooled, excluding the shoulder. This yieldeda sample of 10.3 ml. The protein concentration was estimatedspectrophotometrically by measuring an aliquot of protein diluted25-fold for the measurement, using the following formula:(1.55×OD280-0.76×OD260)×volume. The average concentration was of 1nmol/ml of VLP (2.6 mg/ml). The ratio of absorbance at 280 nm vs. 260 nmwas of 0.12/0.105.

Reassembly: 1.1 ml beta-mercaptoethanol was added to the sample, and thefollowing reassembly reactions were set up:

1 ml of AP205 coat protein, no nucleic acids

1 ml of AP205 coat protein, rRNA (approx. 200 OD260 units, 10 nmol)

9 ml of AP205 coat protein, CyCpG (370 ul of 225 pmol/μl solution, i.e.83 μnmol).

These mixtures were dialyzed 1 hour against 30 ml of NET buffercontaining 10% beta-mercaptoethanol. The mixture containing no nucleicacids was dialyzed separately. The dialysis was then pursued in acontinuous mode, and 1 l of NET buffer was exchanged over 3 days. Thereaction mixtures were subsequently extensively dialyzed against water(5 changes of buffer), and lyophilized. They were resolubilized inwater, and analyzed by electron microscope (EM). All mixtures containedcapsids, showing that AP205 VLP reassembly is independent of thepresence of detectable nucleic acids, as measured by agarose gelelectrophoresis using ethidium bromide staining. The EM procedure was asfollows: A suspension of the proteins was absorbed on carbon-formvarcoated grids and stained with 2% phosphotungstic acid (pH 6,8). Thegrids were examined with a JEM 100C (JEOL,Japan) electron microscope atan accelerating voltage of 80 kV. Photographic records (negatives) wereperformed on Kodak electron image film and electron micrographs wereobtained by printing of negatives on Kodak Polymax paper. The VLPreassembled in the presence of the CyCpG was purified over a Sepharose4B column (1×50 cm), eluted with NET buffer (1 ml/h). The fractions wereanalyzed by Ouchterlony assay, and the fractions containing VLP werepooled. This resulted in a sample of 8 ml, which was desalted againstwater by dialysis, and dried. The yield of capsid was of 10 mg. Analysisof resolubilized material in a 0.6% agarose gel stained withethidium-bromide showed that the capsids were empty of nucleic acids.Samples of the reassembly reaction containing CyCpG taken after thereassembly step and before extensive dialysis were analysed on a 0.6%agarose gel stained with ethidium-bromide and Coomassie blue. A bandmigrating at the same height than intact AP205 VLP and staining both forethidium-bromide and Coomassie blue staining could be obtained, showingthat AP205 VLP containing oligodeoxynucleotide had been reassembled. Theextensive dialysis steps following the reassembly procedure are likelyto have led to diffusion of the oligodeoxynucleotide outside of theVLPs. Significantly, the AP205 VLPs could also be reassembled in theabsence of detectable oligodeoxynucleotide, as measured by agarose gelelectrophoresis using ethidium bromide staining. Oligodeoxynucleotidescould thus be successfully bound to AP205 VLP after initial disassemblyof the VLP, purification of the disassembled coat protein from nucleicacids and subsequent reassembly of the VLP in the presence ofoligodeoxynucleotide.

-   -   B. Reassembly of AP205 VLP using Disassembled Material which        does not Reassemble in the Absence of Added Oligonucleotide

Disassembly: 100 mg of purified and dried recombinant AP205 VLP wereused for disassembly as described above. All steps were performedessentially as described under disassembly in part A, but for the use of8 M urea to solublize the pellets of the ammonium sulphate precipitationsteps and the omission of the gel filtration step using a CL-4B columnprior to reassembly . The pooled fractions of the Sephadex G-75 columncontained 21 mg of protein as determined by spectroscopy using theformula described in part A. The ratio of absorbance at 280 nm to theabsorbance at 260 nm of the sample was of 0.16 to 0.125. The sample wasdiluted 50 times for the measurement.

Reassembly: The protein preparation resulting from the Sephadex G-75 gelfiltration purification step was precipitated with ammonium sulphate at60% saturation, and the resulting pellet solubilized in 2 ml 7 M urea,10 mM DTT. The sample was diluted with 8 ml of 10% 2-mercaptoethanol inNET buffer, and dialyzed for 1 hour against 40 ml of 10%2-mercaptoethanol in NET buffer. Reassembly was initiated by adding 0.4ml of a CyCpG solution (109 nmol/ml) to the protein sample in thedialysis bag. Dialysis in continous mode was set up, and NET buffer usedas eluting buffer. Dialysis was pursued for two days and a sample wastaken for EM analysis after completion of this dialysis step. Thedialyzed reassembly solution was subsequently dialyzed against 50% v/vGlycerol in NET buffer, to achieve concentration. One change of bufferwas effected after one day of dialysis. The dialysis was pursued over atotal of three days.

The dialyzed and concentrated reassembly solution was purified by gelfiltration over a Sepharose 4-B column (1×60 cm) at a flow rate of 1ml/hour, in NET buffer. Fractions were tested in an Ouchterlony assay,and fractions containing capsids were dried, resuspended in water, andrechromatographed on the 4-B column equilibrated in 20 mM Hepes pH 7.6.Using each of the following three formula:(183*OD230 nm−75.8*OD260 nm)*volume (ml)⁻2. ((OD235 nm−OD280nm)/2.51)×volume−3. ((OD228.5 nm−OD234.5 nm)*0.37)×volume   1.

protein amounts of 6-26 mg of reassembled VLP were determined.

The reassembled AP205 VLPs were analyzed by EM as described above,agarose gel electrophoresis and SDS-PAGE under non-reducing conditions.

The EM analysis of disassembled material shows that the treatment ofAP205 VLP with guanidinium-chloride essentially disrupts the capsidassembly of the VLP. Reassembly of this disassembled material with anoligonucleotide yielded capsids, which were purified and furtherenriched by gel filtration. Two sizes of particles were obtained;particles of about 25 nm diameter and smaller particles are visible inthe electron micrograph. No reassembly was obtained in the absence ofoligonucleotides. Loading of the reassembled particles on agaroseelectrophoresis showed that the reassembled particles contained nucleicacids. Extraction of the nucleic acid content by phenol extraction andsubsequent loading on an agarose gel stained with ethidium bromiderevealed that the particles contained the oligonucleotide used forreassembly. Identity of the packaged oligonucleotide was controlled byloading a sample of this oligonucleotide side to side to the nucleicacid material extracted from the particles. The agarose gel where thereassembled AP205 VLP had been loaded and previously stained withethidium bromide was subsequently stained with Coomassie blue, revealingcomigration of the oligonucleotide content with the protein content ofthe particles, showing that the oligonucleotide had been packaged in theparticles. Loaded on the gel were untreated AP205 VLP, 3 samples withdiffering amount of AP205 VLP reassembled with CyCpG and purified, anduntreated Qβ VLP.

Loading of the reassembled AP205 VLP on an SDS-PAGE gel, run in theabsence of reducing agent demonstrated that the reassembled particleshave formed disulfide bridges, as is the case for the untreated AP205VLP. Moreover, the disulfide bridge pattern is identical to theuntreated particles. The samples loaded on the SDS gel were: ProteinMarker, untreated wt Qβ, reassembled wt Qβ, untreated AP205 VLP,reassembled AP205 VLP. The Molecular Weight of the AP205 VLP subunit is14.0 kDa, while the molecular weight of the Qβ subunit is 14.3 kDa (bothmolecular weights calculated with the N-terminal methionine).

-   -   C. Coupling of p33 epitope (sequence: H2N-KAVYNFATMGGCCOOH, with        free N- and C-termini, (SEQ ID NO: 82) to AP205 VLPs reassembled        with CYCpG

Reassembled AP205 VLP obtained as described in part B, and in 20 mMHepes, 150 mM NaCl, pH 7.4 was reacted at a concentration of 1.4 mg/mlwith a 5-fold excess of the crosslinker SMPH diluted from a 50 mM stockin DMSO for 30 minutes at 15° C. The obtained so-called derivatizedAP205 VLP was dialyzed 2×2 hours against at least a 1000-fold volume of20 mM Hepes, 150 mM NaCl, pH 7.4 buffer. The derivatized AP205 wasreacted at a concentration of 1 mg/ml with either a 2.5-fold, or with a5-fold excess of peptide, diluted from a 20 mM stock in DMSO, for 2hours at 15° C. The sample was subsequently flash frozen in liquidnitrogen for storage.

The coupling reaction was analyzed on an SDS-PAGE. Loaded on a gel werethe following samples: protein marker; derivatized AP205 VLP (d); AP205VLP coupled with a 2.5-fold excess of peptide, supernatant (s); AP205VLP coupled with a 2.5-fold excess of peptide, pellet (p); AP205 VLPcoupled with a 5-fold excess of peptide, supernatant (s); AP205 VLPcoupled with a 5-fold excess of peptide, pellet (p). The result of thecoupling reaction revealed that a higher degree of coupling could beachieved by using a 5-fold excess of peptide rather than with a 2.5 foldexcess of peptide in the coupling reaction.

EXAMPLE 11

Non-enzymatic hydrolysis of the RNA content of VLPs and packaging ofimmunostimulatory nucleic acids.

ZnSO4 dependent degradation of the nucleic acid content of a VLP:

5 mg Qβ VLP (as determined by Bradford analysis) in 20 mM HEPES, pH 7.4,150 mM NaCl was dialysed either against 2000 ml of 50 mM TrisHCl pH 8.0,50 mM NaCl, 5% glycerol, 10 mM MgCl2 or 2000 ml of 4 mM HEPES, pH 7.4,30 mM NaCl for 2 h at 4° C in SnakeSkin™ pleated dialysis tubing(Pierce, Cat. No. 68035). Each of the dialysis buffers was exchangedonce and dialysis was allowed to continue for another 16 h at 4° C. Thedialysed solution was clarified for 10 minutes at 14 000 rpm (Eppendorf5417 R, in fixed angle rotor F45-30-11, used in all the following steps)and Protein concentration was again determined by Bradford analysis. QβVLPs in 50 mM TrisHCl pH 8.0, 50 mM NaCl, 5% glycerol, 10 mM MgCl2 werediluted with the corresponding buffer to a final protein concentrationof 1 mg/ml whereas Qβ VLPs in 4 mM HEPES pH 7.4, 30 mM NaCl were dilutedwith the corresponding buffer to a final protein concentration of 0.5mg/ml. This capsid-containing solutions were centrifuged again for 10minutes at 14 000 rpm at 4° C. The supernatants were than incubated withZnSO4 which was added to a final concentration of 2.5 mM for 24 h at 60°C. in an Eppendorf Thermomixer comfort at 550 rpm. After 24 h thesolutions were clarified for 10 minutes at 14000 rpm and the sedimentwas discarded. The efficiency of the ZnSO4-dependent degradation ofnucleic acids was confirmed by agarose gelelectrophoresis. Thesupernatants were dialysed against 5000 ml of 4 mM HEPES pH 7.4, 30 mMNaCl for 2 h at 4° C. 5000 ml buffer was exchanged once and dialysiscontinued over night at 4° C. The dialysed solution was clarified for 10minutes at 14 000 rpm and 4° C., a negligible sediment was discarded andthe protein concentration of the supernatants were determined byBradford analysis. Similar results were obtained with copperchloride/phenanthroline/hydrogen peroxide treatment of capsids. Thoseskilled in the art know alternative non-enzymatic procedures forhydrolysis or RNA.

ZnSO4-treated Qβ VLPs was analyzed by agarose gelelectrophoresis: QβVLPs which had been purified from E. coli and dialysed either againstbuffer 1 (50 mM TrisHCl pH 8.0, 50 mM NaCl, 5% glycerol, 10 mM MgCl2) orbuffer 2 (4 mM HEPES, pH 7.4, 30 mM NaCl) were incubated either withoutor in the presence of 2.5 mM zinc sulfate (ZnSO4) for 24 hrs at 60° C.After this treatment equal amounts of the indicated samples (5 μgprotein) were mixed with loading dye and loaded onto a 0.8% agarose gel.After the run the gel was stained with ethidium bromide. Treatment ofVLPs with ZnSO4 caused degradation of the nucleic acid content, whilethe mock-treated controls were unaffected.

Packaging of Oligodeoxynucleotides into ZnSO4-Treated VLPs:

ZnSO4-treated and dialysed Qβ capsids with a protein concentration (asdetermined by Bradford analysis) beween 0.4 mg/ml and 0.9 mg/ml (whichcorresponds to a concentration of capsids of 159 nM and 357.5 nM,respectively) were used for the packaging of the oligodeoxynucleotides.The oligodeoxynucleotides were added at a 300-fold molar excess to theof Qβ-VLP capsids and incubated for 3 h at 37° C. in an EppendorfThermomixer comfort at 550 rpm . After 3 h the reactions werecentrifuged for 10 minutes at 14 000 rpm and 4° C. The supernatants weredialysed in Spectra/Por®CE DispoDialyzer with a MWCO 300,000 (Spectrum,Cat. No. 135 526) against 5000 ml of 20 mM HEPES pH 7.4, 150 mM NaCl for8 h at 4° C. 5000 ml buffer was exchanged once and dialysis continuedover night at 4° C. The protein concentration of the dialysed sampleswere determined by Bradford analysis. Qβ capsids and their nucleic acidcontents were analyzed as described in Examples 7 and 9.

Packaging of oligodeoxynucleotides into ZnSO4-treated VLPs was analyzedby agarose gelelectrophoresis. Qβ VLPs which had been treated with 2.5mM zinc sulfate (+ZnSO4) were dialysed against 4 mM HEPES, pH 7.4, 30 mMNaCl and incubated for 3 hrs at 37° C. with an excess ofoligodeoxynucleotides (due to the dialysis the concentration of ZnSO4was decreased by an order of 106, therefore its indicated only inparenthesis) After this incubation in presence of oligodeoxynucleotides,equal amounts of the indicated samples (5 μg protein) were mixed withloading dye and loaded onto a 0.8% agarose gel. After the run the gelwas stained with ethidium bromide. Adding of oligodeoxynucleotides toZnSO4-treated Qβ VLPs could restore the electrophoretical behaviour ofthe so treated capsids when compared to untreated Qβ capsids which hadbeen purified from E. coli.

The nucleic acid content of ZnSO4- and oligodeoxynucleotide treated QβVLPs was analyzed by Benzonase and proteinase K digestion andpolyacrylamide TBE/Urea gelelectrophoresis: Oligodeoxynucleotides werepackaged into ZnSO4-treated Qβ VLPs as described above. 25 μg of theseVLPs were digested with 25 μl Benzonase (Merck, Cat. No. 1.01694.0001)according to the manufactures instructions. After heat-inactivation ofthe nuclease (30 minutes at 80° C.) the VLPs were treated withProteinase K (final enzyme concentration was 0.5 mg/ml) according to themanufactures instructions. After 3 hrs the equivalent of 2 ug Qβ VLPswhich had been digested by Benzonase and proteinase K were mixed withTBE-Urea sample buffer and loaded on a 15% polyacrylamide TBE-Urea gel(Novex®, Invitrogen Cat. No. EC6885). The capsids loaded in lane 2 weretreated with 2.5 mM ZnSO4 in presence of buffer 1 (see above), while thecapsids loaded in lane 3 were treated with 2.5 mM ZnSO4 in presence ofbuffer 2 (see above). As qualitative as well as quantitative standard, 1pmol, 5 pmol and 10 pmol of the oligodeoxynucleotide which was used forthe reassembly reaction, was loaded onto the same gel (lanes 4 -6). Ascontrol, Qβ capsids which had been purified from E. coli were treatedexactly the same and analyzed on the same polyacrylamide TBE-Urea gel(lane 1). After the run was completed, the gel was fixed, equilibratedto neutral pH and stained with SYBR-Gold (Molecular Probes Cat. No.S-11494). Intact Qβ VLPs (which had been purified from E. coli) did notcontain nucleic acids of similar size than those which had beenextracted from ZnSO4-and oligodeoxynucleotide treated Qβ capsids. Inaddition, nucleic acids isolated from the latter VLPs were comigratingwith the oligodeoxynucleotides which had been used in the reassemblyreaction. This results confirmed that the used oligodeoxynucleotideswere packaged into ZnSO4-treated Qβ capsids.

EXAMPLE 12

Coupling of Antigenic Peptides after Packaging of ImmunostimulatoryNucleic Acids into VLPs.

RNaseA and ZnSO4 mediated degradation of the nucleic acid content of aVLP.

Qβ VLPs were treated with RNaseA as described in Example 9 under lowionic strength conditions (20 mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl,pH 7.4 ). Similarly, other VLPs such as described in Examples 2, 3, 7,and 10, i.e. GA, BKV, HBcAg, and AP205 are treated. Alternatively, QβVLPs and AP205 VLPs were treated with ZnSO4 under low ionic strengthconditions (20 mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl pH 7.4) asdescribed in Example 11. AP205 VLP (1 mg/ml) in either 20 mM Hepes pH7.4 or 20 mM Hepes, 1 mM Tris, pH 7.4 was treated for 48 h with 2.5 mMZnSO4 at 50° C. in an Eppendorf Thermomixer comfort at 550 rpm. Qβ andAP205 VLP samples were clarified as described in Example 11 andsupernatants were dialysed in 10.000 MWCO Spectra/Por® dialysis tubing(Spectrum, Cat. nr. 128 118) against first 2 l 20 mM Hepes, pH 7.4 for 2h at 4° C. and, after buffer exchange, overnight. Samples were clarifiedafter dialysis as described in Example 11 and protein concentration inthe supernatants was determined by Bradford analysis.

Packaging of ISS into RnaseA and ZnSO4 Treated VLPs.

After RNA hydrolysis and dialysis, Qβ and AP205 VLPs (1-1.5 mg/ml) weremixed with 130 μl of CpG oligonucleotides (NKCpG, G10-PO—cf. Table 2;G3-6, G8-8—cf. Table 3; 1 mM oligonucleotide stock in 10 mM Tris pH 8)per ml of VLPs. Samples were incubated for 3 h at 37° C. in athermoshaker at 650 rpm. Subsequently, samples were treated with 125 UBenzonase/ml VLPs (Merck KGaA, Darmstadt, Germany) in the presence of 2mM MgCl2 and incubated for 3 h at 37° C. before dialysis. Samples weredialysed in 300.000 MWCO Spectra/Por® dialysis tubing (Spectrum, Cat.nr. 131 447) against 20 mM Hepes, pH 7.4 for 2 h at 4° C., and afterbuffer exchange overnight against the same buffer. After dialysissamples were clarified as described in Example 11 and proteinconcentration in the supernatants were determined by Bradford analysis.

Coupling of Immunogenic Peptides to ISS Packaged VLPs.

Qβ VLPs, packaged with ISS were coupled to p33 peptides containing aC-terminal GGC extension (KAVYNFATM-GGC) (SEQ ID NO: 82), resulting inQb VLPs termed Qb-ISS-33 VLPs. Packaged Qβ VLPs in 20 mM Hepes, pH 7.4were derivatized with a 10-fold molar excess of SMPH (Pierce) for 0.5 hat 25° C., followed by two dialysis steps of 2 hours each against 20 mMHEPES pH 7.4 at 4° C. to remove unreacted SMPH. Peptides were added in a5-fold molar excess to the dialysed derivatization mixture, and allowedto react for 2 h in a thermomixer at 25° C. Samples were dialysed in300.000 MWCO Spectra/Por® dialysis tubing against 20 mM Hepes pH 7.4 for2 h at 4° C., and after buffer exchange overnight against the samebuffer. After dialysis samples were clarified as described in Example 11and protein concentration in the supernatants were determined byBradford analysis. Coupling of peptide p33 to Qβ was analysed bySDS-PAGE on 16% PAGE Tris-Glycine gels (Novex® by Invitrogen, Cat. No.EC64952), using a sample buffer containing 2% SDS and β-mercapto ethanolor DTT. Packaging was analysed on 1% agarose gels and, after proteinaseK digestion, on TBE/urea gels as described in Example 7.

AP205 VLPs (1.24 mg/ml) packaged with G8-8 oligonucleotide as describedabove are derivatized and coupled to MelanA 16-35 A/L containing aN-terminal C extension (c GHGHSYTTAE ELAGIGILTV) (SEQ ID NO: 55),resulting in AP205-G8-8-MelanA VLPs. AP205 VLPs (packaged with G8-8), in20 mM Hepes pH 7.4, are derivatized with a 20-fold molar excess of SMPHfor 0.5 h at 25° C., and subsequently dialysed two times against 20 mMHEPES, pH 7.4 at 4° C. to remove unreacted SMPH. Peptide is added to thedialyzed derivatization mixture in a 10-fold molar excess and allowed toreact for 2 h in a thermomixer at 25° C. Samples were dialysed in 10.000MWCO dialysis tubing against 20 mM Hepes pH 7.4 for 2 h at 4° C., andafter buffer exchange, overnight against the same buffer. Afterdialysis, samples are clarified as described in Example 11 and proteinconcentration in the supernatants are determined by Bradford analysis.Coupling efficiency of peptide MelanA 16-35 A/L to AP205 is analysed bySDS-PAGE on 16% PAGE Tris-Glycine gels. G8-8 oligonucleotide packagingin AP205 is analysed on 1% agarose gels and, after proteinase Kdigestion, G8-8 oligonucleotide amount in AP205-G8-8- MelanA 16-35 A/Lis analysed on TBE/urea gels as described in Example 7.

Packaging of RNAseA and ZnSO4-treated Qβ VLPs with NKCpG before as wellas after coupling to p33 peptide was analyzed by agarosegelelectrophoresis. Qβ VLPs containing NKCpG oligonucleotides andsubsequently coupled to p33 peptide were termed Qb-NKCpG-33 VLPs. On a1% agarose gel, the fluorescent band visible on the ethidium bromidestained gel co-migrates with the protein band visible on the CoomassieBlue stained gel demonstrating packaging. Thus, upon packaging, bothRNaseA and ZnSO4 treated Qβ VLPs contain NKCpG oligonucleotides beforeas well as after coupling to p33 peptide. Coupling efficiency of the p33peptide is maintained as can be judged from the multiple couplingproducts visible after SDS-PAGE analysis on a 16% PAGE Tris-Glycine gel,as bands migrating slower than residual Qβ VLP subunit monomers whichhave not reacted with peptide. The packaging efficiency can be estimatedfrom the analysis of the TBE/urea gel by comparison of the signal of theoligonucleotide from the packaged Qb-NKCpG-33 lane with the signal ofthe oligonucleotide standard loaded on the same gel. Packaged amounts ofNKCPG were between 1 and 4 nmol/100 μg Qb-NKCpG-33 VLPs.

Packaging of G8-8 oligonucleotides into Qβ VLPs and subsequent couplingto p33 peptide was analyzed by agarose gelelectrophoresis. Qβ VLPscontaining G8-8 oligonucleotides and subsequently coupled to p33 peptidewere termed Qb-G8-8-33 VLPs. Ethidium bromide staining of G8-8 packagedQβ VLPs can be seen on a 1% agarose gel stained with ethidium bromide.Comigration of the ethidium bromide fluorescent band with the Qβ VLPprotein band visible on the same gel subsequently stained with CoomassieBlue demonstrates packaging. Coupling efficiency can be estimated to be30% by SDS-PAGE analysis on a 16% PAGE Tris-Glycine gel. Analysis of theG8-8 content of Qb-G8-8-33 VLPs was done on a 1% agarose gel, where theamount of oligonucleotide packaged was of approximately 1 nmol/100 μgQb-G8-8-33 VLPs.

Packaging of G8-8 oligonucleotides into AP205 VLPs was analyzed byagarose gelelectrophoresis. Staining of G8-8 packaged AP205 VLPs can beseen on a 1% agarose gel stained with ethidium bromide. Comigration ofthe AP205 VLPs protein band detected on the same gel subsequentlystained with Coomassie Blue demonstrated packaging. Coupling efficiencywith the MelanA 16-35 A/L peptide can be estimated from the SDS-PAGEanalysis on a 16% PAGE Tris-Glycine gel where multiple coupling bandsmigrating slower than the residual AP205 VLP monomer subunits, which didnot react with peptide, cam be visible. Coupling efficiency is comparedto the coupling efficiency obtained for the Qb-G8-8-33 VLPs. Analysis ofthe G8-8 oligonucleotide content of AP205 VLPs after coupling to MelanA16-35 A/L can be seen on TBE/urea gel electrophoresis.

EXAMPLE 13

Packaging of Immunostimulatory Guanosine Flanked Oligonucleotides intoVLPs.

Qbx33 VLPs (Qβ VLPs coupled to peptide p33, see Example 9) were treatedwith RNaseA under low ionic conditions (20 mM Hepes pH 7.4) as describedin Example 9 to hydrolyse RNA content of the Qbx33 VLP. After dialysisagainst 20 mM Hepes pH 7.4, Qbx33 VLPs were mixed with guanosine flankedoligonucleotides (Table 2: G10-PO, Table 3: G3-6, G7-7, G8-8, G9-9 orG6, from a 1 mM oligonucleotide stock in 10 mM Tris pH 8) and incubatedas described in Example 12. Subsequently, Qbx33 VLPs were treated withBenzonase and dialysed in 300.000 MWCO tubing. Samples with oligos G7-7,G8-8 and G9-9 were extensively dialysed over 3 days with 4 bufferexchanges to remove free oligo. Packaging was analysed on 1% agarosegels and, after proteinase K digestion, on TBE/urea gels as described inExample 7.

TABLE 3 Sequences of immunostimulatory nucleic acids used in theExamples. ISS name 5′-3′ sequence SEQ ID NO GACGATCGTC 1 G3-6GGGGACGATCGTCGGGGGG 2 G4-6 GGGGGACGATCGTCGGGGGG 3 G5-6GGGGGGACGATCGTCGGGGGG 4 G6-6 GGGGGGGACGATCGTCGGGGGG 5 G7-7GGGGGGGGACGATCGTCGGGGGGG 6 G8-8 GGGGGGGGGACGATCGTCGGGGGGGG 7 G9-9GGGGGGGGGGACGATCGTCGGGGGGGGG 8 G6 GGGGGGCGACGACGATCGTCGTCGGGGGGG 9G10-PO GGGGGGGGGGGACGATCGTCGGGGGGGGGG 41

Packaging of G3-6, G6, G8-8 oligonucleotides in RNaseA treated Qbx33VLPs was analyzed by agarose gelelectrophoresis. Upon oligonucleotidepackaging, a fluorescent band migrating slightly slower than referenceuntreated Qβ VLP becomes visible on the 1% agarose gel stained withethidium bromide indicating the presence of oligonucleotides. The signalis maintained after treatment with Benzonase, indicating packaging ofthe oligonucleotides within the Qbx33 VLPs. The packaging efficiency canbe estimated from the TBE/urea gel electrophoresis. The amount of theG3-6 oligonucleotide (approximately 4 nmol/100 μg Qbx33 VLPs) packagedis much higher than the amount of packaged G8-8 oligonucleotide(approximately 1 nmol/100 μg Qbx33 VLPs). This indicates a dependence ofpackaging ability on the length of the guanosine nucleotides tailflanking the CpG motif.

EXAMPLE 14

Packaging Ribonucleic Acid into VLPs.

ZnSO4 dependent degradation of the nucleic acid content of a VLP.

Qβ VLPs were treated with ZnSO4 under low ionic strength conditions (20mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl, pH 7.4) as described inExample 11. AP205 VLPs (1 mg/ml) in either 20 mM Hepes pH 7.4 or 20 mMHepes, 1 mM Tris, pH 7.4 were treated for 48 h with 2.5 mM ZnSO4 at 50°C. in an Eppendorf Thermomixer comfort at 550 rpm. Qβ and AP205 VLPsamples were clarified as in Example 11 and dialysed against 20 mMHepes, pH 7.4 as in Example 12.

Packaging of Poly (I:C) into ZnSO4-Treated VLPs:

The immunostimulatory ribonucleic acid poly (I:C), (Cat. nr. 27-4732-01,poly(I).poly(C), Pharmacia Biotech) was dissolved in PBS (Invitrogencat. nr. 14040) or water to a concentration of 4 mg/ml (9 μM). Poly(I:C) was incubated for 10 minutes at 60° C. and then cooled to 37° C.Incubated poly (I:C) was added in a 10-fold molar excess to eitherZnSO4-treated Qβ p or AP205 VLPs (1-1.5 mg/ml) and the mixtures wereincubated for 3 h at 37° C. in a thermomixer at 650 rpm. Subsequently,excess of free poly (I:C) was enzymatically hydrolysed by incubationwith 125 U Benzonase per ml VLP mixture in the presence of 2 mM MgCl2for 3 h at 37° C. in a thermomixer at 300 rpm. Upon Benzonase hydrolysissamples were clarified as described in Example 11 and supernatants weredialysed in 300.000 MWCO Spectra/Por® dialysis tubing (Spectrum, Cat.nr. 131 447) against 2 120 mM Hepes, pH 7.4 for 2 h at 4° C., and afterbuffer exchange overnight against the same buffer.. After dialysis,samples were clarified as described in Example 11 and proteinconcentration in the supernatants were determined by Bradford analysis.

Coupling of Immunogenic Peptides to Poly (I:C) Packaged VLPs.

Qβ VLPs (1 mg/ml) packaged with poly (I:C) were derivatized and coupledeither to p33 peptide (KAVYNFATM-GGC) (SEQ ID NO: 82) as described inExample 12, or to MelanA peptide (MelanA 16-35A/L CGHGHSYTTAEELAGIGILTV)(SEQ ID NO: 55), resulting in Qb-pIC-33 and Qb-pIC-MelanA VLPs,respectively. For coupling to MelanA peptide, the packaged Qβ VLP wasderivatized with a 2.1 -fold molar excess of SMPH (Pierce) for 0.5 h at25° C., followed by two dialysis steps against 20 mM HEPES, pH 7.4 at 4°C. to remove unreacted SMPH. Peptides were added in a 2.1 -fold molarexcess and allowed to react for 1.5 h in a thermomixer at 25° C. Sampleswere dialysed in 300.000 MWCO Spectra/Por® CE Dispo Dialyzer against 20mM Hepes, pH 7.2 for 3 h at 4° C., and after buffer exchange, overnightagainst the same buffer. After dialysis samples were clarified asdescribed in Example 11 and protein concentration in the supernatantswere determined by Bradford analysis. Coupling of peptide p33 andpeptide MelanA to Qβ was analysed by SDS-PAGE on 16% PAGE Tris-Glycinegels. Packaging was analysed on 1% agarose gels and, after proteinase Kdigestion, on TBE/urea gels as described in Example 7.

Packaging of poly (I:C) into ZnSO4 treated Qβ VLPs and coupling withMelanA peptide resulting in Qb-pIC-MelanA VLPs was analyzed by agarosegelelectrophoresis. The fluorescent signal visible on an ethidiumbromide stained 1% agarose gel, indicating presence of nucleic acid,co-migrates with the protein band that became visible upon CoomassieBlue staining of the gel, demonstrating packaging. Coupling efficiencyof the MelanA peptide was estimated by SDS-PAGE analysis on a 16% PAGETris-Glycine gel. Multiple coupling products were visible as bandsmigrating slower than the Qβ VLP monomer subunits, which had not reactedwith peptide. Coupling efficiency of MelanA was overall comparable tothe coupling efficiency obtained for the Qb-G8-8-33 VLPs (Example 12),albeit slightly lower. The packaging efficiency into Qb-pIC-MelanA couldbe estimated from the TBE/urea gel; the packaged amount of poly (I:C) inQβ was approximately 25 pmol and remained the same upon MelanA coupling.

AP205 VLPs (1 mg/ml) packaged with poly (I:C) are derivatized andcoupled to MelanA 16-35 A/L containing a N-terminal C extension(cGHGHSYTTAE ELAGIGILTV) (SEQ ID NO: 55), resulting in AP205-G8-8-MelanAVLPs. AP205 VLPs, in 20 mM Hepes, pH 7.4 are derivatized with a 20-foldmolar excess of SMPH for 0.5 h at 25° C., and subsequently dialysed twotimes against 20 mM HEPES, pH 7.4 at 4° C. to remove unreacted SMPH.Peptide is added to the dialyzed derivatization mixture in a 10-foldmolar excess and allowed to react for 2 h in a thermomixer at 25° C.Samples are dialysed in 10.000 MWCO dialysis tubing against 20 mM HepespH 7.4 for 2 h at 4° C., and after buffer exchange, overnight againstthe same buffer. After dialysis, samples are clarified as described inExample 11 and protein concentration in the supernatants are determinedby Bradford analysis. Coupling efficiency of peptide MelanA 16-35 A/L toAP205 is analysed by SDS-PAGE on 16% PAGE Tris-Glycine gels. Poly (I:C)packaging is analysed on 1% agarose gels and, after proteinase Kdigestion, on TBE gels as described in Example 7.

Packaging of poly (I:C) into ZnSO4 treated AP205 VLPs and the couplingproduct AP205-pIC-MelanA after coupling to MelanA is analyzed by agarosegelelectrophoresis. Coupling efficiency of the MelanA peptide isestimated from the appearance of multiple coupling products visible asbands migrating slower than AP205 VLP subunit monomer, which do notreact with peptide, after SDS-PAGE analysis on a 16% PAGE Tris-Glycinegel electrophoresis. The packaging efficiency can be estimated from theTBE gel.

EXAMPLE 15

Packaging of Immunostimulatory Guanosine Flanked Oligonucleotides intoHBcAg VLPs.

HBcAg VLPs are treated with RNaseA under low ionic strength conditions(20 mM Hepes pH 7.4) as described in Example 9 to hydrolyse RNA contentof the VLP. After dialysis against 20 mM Hepes, pH 7.4, VLPs are mixedwith guanosine flanked oligonucleotides (Table 3; G3-6, G7-7, G8-8,G9-9, G10-PO or G6, 1 mM stock in 10 mM Tris pH 8) and incubated asdescribed in Example 12. Subsequently, VLPs are treated with Benzonaseand dialysed in 300.000 MWCO tubing. Packaging is analysed on 1% agarosegels and on TBE/urea gels after proteinase K digestion as described inExample 7.

EXAMPLE 16

Packaging of Immunostimulatory Guanosine Flanked Oligonucleotides intoGA VLPs.

GA VLPs are treated with RNaseA under low ionic conditions (20 mM HepespH 7.4) as described in Example 9 to hydrolyse RNA content of the VLP.After dialysis against 20 mM Hepes pH 7.4, VLPs are mixed with guanosineflanked oligonucleotides (Table 3; G3-6, G7-7, G8-8, G9-9, G10-PO or G6,1 mM stock in 10 mM Tris pH8) and incubated as described in Example 12.Subsequently, VLPs are treated with Benzonase and dialysed in 300.000MWCO tubing. Packaging is analysed on 1% agarose gels and on TBE/ureagels after proteinase K digestion as described in Example 7.

EXAMPLE 17

Packaging Ribonucleic Acid into HBcAg VLPs.

HBcAg VLPs are treated with ZnSO4 under low ionic strength conditions(20 mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl, pH 7.4 ) as described inExample 11 and are dialysed against 20 mM Hepes pH 7.4 as in Example 12.Poly (I:C) is added in a 10-fold molar excess to HBcAg VLPs (1-1.5mg/ml) and incubated for 3 h at 37° C. in a thermomixer at 650 rpm asdescribed in Example 14. Subsequently, excess of free poly (I:C) isenzymatically hydrolysed by incubation with 125 U Benzonase per ml VLPmixture in the presence of 2 mM MgCl2 for 3 h at 37° C. in a thermomixerat 300 rpm. Samples are clarified after Benzonase hydrolysis asdescribed in Example 11 and dialysed as in Example 14. After dialysis,samples are clarified as described in Example 11 and proteinconcentration in the supernatants are determined by Bradford analysis.HBcAg VLPs (1 mg/l) packaged with poly (I:C) are derivatized and coupledto MelanA, and dialysed as in Example 14.

EXAMPLE 18

Packaging Ribonucleic Acid into GA VLPs.

GA VLPs are treated with ZnSO4 under low ionic strength conditions (20mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl, pH 7.4 ) as described inExample 11 and are dialysed against 20 mM Hepes, pH 7.4 as in Example12. Poly (I:C) is added in a 10-fold molecular excess to GA VLPs (1-1.5mg/ml) and incubated for 3 h at 37° C. in a thermomixer at 650 rpm asdescribed in Example 14. Subsequently, excess of free poly (I:C) isenzymatically hydrolysed by incubation with 125 U Benzonase per ml VLPmixture in the presence of 2 mM MgCl2 for 3 h at 37° C. in a thermomixerat 300 rpm. Samples are clarified after Benzonase hydrolysis asdescribed in Example 11 and dialysed as in Example 14. After dialysis,samples are clarified as described in Example 11 and proteinconcentration in the supernatants are determined by Bradford analysis.GA VLPs (1 mg/ml) packaged with poly (I:C) are derivatized and coupledto MelanA, and dialysed as in Example 14.

EXAMPLE 19

Qβ Disassembly, Reassembly and Packaging of Oligodeoxynucleotides.

Disassembly and Reassembly of Qβ VLP

Disassembly: 45 mg Qβ VLP (2.5 mg/ml, as determined by Bradfordanalysis) in PBS (20 mM Phosphate, 150 mM NaCl, pH 7.5), was reducedwith 10 mM DTT for 15 min at RT under stirring conditions. A secondincubation of 15 min at RT under stirring conditions followed afteraddition of magnesium chloride to a final concentration of 700 mM,leading to precipitation of the encapsulated host cell RNA andconcomitant disintegration of the VLPs. The solution was centrifuged 10min at 4000 rpm at 4° C. (Eppendorf 5810 R, in fixed angle rotor A-4-62used in all following steps) in order to remove the precipitated RNAfrom the solution. The supernatant, containing the released, dimeric Qβcoat protein, was used for the chromatography purification steps.

Two-step purification method for Qβ coat protein by cation exchangechromatography and size exclusion chromatography: The supernatant of thedisassembly reaction, containing dimeric coat protein, host cellproteins and residual host cell RNA, was applied onto a SP-Sepharose FFcolumn (xk16/20, 6 ml, Amersham Bioscience). During the run, which wascarried out at RT with a flow rate of 5 ml/min, the absorbance at 260 nmand 280 nm was monitored. The column was equilibrated with 20 mM sodiumphosphate buffer pH 7 and the sample was diluted 1:15 in water to adjusta conductivity below 10 mS/cm in order to achieve proper binding of thecoat protein to the column. The elution of the bound coat protein wasaccomplished by a step gradient to 20 mM sodium phosphate/500 mM sodiumchloride and the protein was collected in a fraction volume of approx.25 ml. The column was regenerated with 0.5 M NaOH.

In the second step, the isolated Qβ coat protein dimer (the elutedfraction from the cation exchange column) was applied (in two runs) ontoa Sephacryl S-100 HR column (xk26/60, 320 ml, Amersham Bioscience)equilibrated with 20 mM sodium phosphate/250 mM sodium chloride; pH 6.5.Chromatography was performed at RT with a flow rate of 2.5 ml/min.Absorbance was monitored at 260 nm and 280 nm. Fractions of 5 ml werecollected. The column was regenerated with 0.5 M NaOH.

Reassembly by dialysis: A stock solution of purified Qβ coat proteindimer at a concentration of 2 mg/ml was used for the reassembly of QβVLP in the presence of the oligodeoxynucleotide G8-8 or G10-PO. Theconcentration of oligodeoxynucleotide in the reassembly mixture was 10μM. The concentration of coat protein dimer in the reassembly mixturewas 40 μM (approx. 1.13 mg/ml). Stock solutions of urea and DTT wereadded to the solution to give final concentrations of 1 M urea and 5 mMDTT respectively. The oligodeoxynucleotide was added as last component,together with H₂O, giving a final volume of the reassembly reaction of 3ml. This solution was dialysed at 4° C. for 72 h against 1500 ml buffercontaining 20 mM TrisHCl, 150 mM NaCl, pH 8.0. The dialysed reassemblymixture was centrifuged at 14 000 rpm for 10 minutes at 4° C. Anegligible sediment was discarded while the supernatant contained thereassembled and packaged VLPs. Reassembled and packaged VLPs wereconcentrated with centrifugal filter devices (Millipore, UFV4BCC25, 5KNMWL) to a final protein concentration of 3 mg/ml. Protein concentrationwas determined by Bradford analysis.

Purification of reassembled and packaged VLPs by size exclusionchromatography: Up to 10 mg total protein was loaded onto a Sepharose™CL-4B column (xk16/70, Amersham Biosciences) equilibrated with 20 mMHEPES, 150 mM NaCl, pH 7.4. The chromatography was performed at roomtemperature at a flow-rate of 0.4 ml/min. Absorbance was monitored at260 nm and 280 nm. Two peaks were observed, collected in fractions of0.5 ml size and analysed by SDS-PAGE. The disulfide-bond pattern inreassembled and purified Qβ capsids was analyzed by non-reducingSDS-PAGE. 5 μg of the indicated capsids were mixed with sample buffer(containing SDS) that contained no reducing agent and loaded onto a 16%Tris-Glycine gel. After the run was completed the gel was stained withCoomassie blue. When compared to “intact” capsids purified from E. coli,the reassembled Qβ VLP displayed the same disulfide bond pattern withthe bands corresponding to dimer, trimer, tetramer, pentamer andhexamers of the Qb coat protein. Calibration of the column with intactand highly purified Qβ capsids from E. coli revealed that the apparentmolecular weight of the major first peak was consistent with Qβ capsids.

Reassembly by diafiltration (optimized method): 20 ml of a stocksolution of purified coat protein (1.5 mg/ml) was mixed with stocksolutions of urea, DTT, oligodeoxynucleotide G10-PO and water. Theoligodeoxynucleotide was added as last component. The volume of themixture was 30 ml and the final concentrations of the components are 35μM dimeric coat protein (reflecting 1 mg/ml), 35 μgoligodeoxynucleotide, 1 M urea and 2.5 mM DTT. The mixture was thendiafiltrated against 300 ml of 20 mM sodium phosphate/250 mM sodiumchloride, pH 7.2, in a tangential flow filtration apparatus at RT, usinga Pellicon XL membrane cartridge (Biomax 5K, Millipore). The total flowrate was set to 10 ml/min and the permeate flow rate set to 2.5 ml/min.After completion of the diafiltration step, H₂O₂ was added to thesolution to a final concentration of 7 mM and the solution was furtherincubated at RT for 60 min, to accelerate the formation of thestructural disulfide bonds in the formed VLPs. The removal ofnon-incorporated oligodeoxynucleotide and coat protein was achieved by a2^(nd) diafiltration against 600 ml of 20 mM sodium phosphate/250 mMsodium chloride, pH 7.2, using a Pellicon XL membrane cartridge (PLCMK300K, Millipore).

Analysis of Qβ VLPs which had been reassembled in the presence ofoligodeoxynucleotides:

A) Hydrodynamic size of reassembled capsids: Qβ capsids, which had beenreassembled in the presence of oligodeoxynucleotide G8-8, were analyzedby dynamic light scattering (DLS) and compared to intact Qβ VLPs, whichhad been purified from E. coli. Reassembled capsids showed the samehydrodynamic size (which depends both on mass and conformation) as theintact Qβ VLPs.

B) Disulfide-bond formation in reassembled capsids: Reassembled Qβ VLPswere analyzed by non-reducing SDS-PAGE and compared to intact Qβ VLPs,which had been purified from E. coli. Reassembled capsids displayed aband pattern, with the presence of disulfide-linked pentameric andhexameric forms of the coat protein, similar to the intact Qβ VLPs (asdescribed above).

C) Analysis of nucleic acid content of the Qβ VLPs which had beenreassembled in the presence of oligodeoxynucleotides by denaturingpolyacrylamide TBE-Urea gelelectrophoresis: Reassembled Qβ VLPs (0.4mg/ml) containing G8-8 oligodeoxynucleotides were incubated for 2 h at37° C. with 125 U benzonase per ml Qβ VLPs in the presence of 2 mMMgCl₂. Subsequently the benzonase treated Qβ VLPs were treated withproteinase K (PCR-grade, Roche Molecular Biochemicals, Cat. No. 1964364)as described in Example 7. The reactions were then mixed with a TBE-Ureasample buffer and loaded on a 15% polyacrylamide TBE-Urea gel (Novex®,Invitrogen Cat. No.

EC6885). As a qualitative as well as quantitative standard, 1 pmol, 5pmol and 10 pmol of the oligodeoxynucleotide which was used for thereassembling reaction, was loaded on the same gel. This gel was stainedwith SYBR®-Gold (Molecular Probes Cat. No. S-11494). The SYBR®-Goldstain showed that the reassembled Qβ capsids contained nucleic acidco-migrating with the oligodeoxynucleotides which were used in thereassembly reaction. Taken together, resistance to benzonase digestionof the nucleic acid content of the Qβ VLPs which had been reassembled inthe presence of oligodeoxynucleotides and isolation of theoligodeoxynucleotide from purified particles by proteinase K digestion,demonstrate packaging of the oligodeoxynucleotide.

EXAMPLE 20

Coupling of Peptides Derived from MelanA Melanoma Antigen to Qb

TABLE 4 The following MelanA peptide moieties were chemicallysynthesized: SEQ ID Abbreviation* Sequence** NO: ELAGIGILTV 50GHGHSYTTAE ELAGIGILTV 51 SYTTAEELAGIGILTV ILGVL 52 ELAGIGILTVILGVL 53MelanA 16-35 c GHGHSYTTAE EAAGIGILTV 54 MelanA 16-35 A/L c GHGHSYTTAEELAGIGILTV 55 MelanA 26-35 cgg EAAGIGILTV 56 MelanA 26-35 A/L cggELAGIGILTY 57 MelanA 20-40 A/L c SYTTAEELAGIGILTV ILGVL 58 MelanA 26-40A/L cgg ELAGIGILTVILGVL 59 MelanA 26-35-C A/L ELAGIGILTV ggc 60CSPKSL-MelanA 26-35 CSPKSLELAGIGILTV 92 A/L MelanA 26-40-C A/LELAGIGILTVILGVLGGC 93 *A/L indicates alanin to lysine exchange comparedto the original wildtype MelanA peptide **amino acids from the linkersequence are indicated in small letters

The following procedures were used for chemical coupling of the MelanApeptide moieties to Qb VLPs:

For peptide MelanA 16-35, MelanA 16-35 A/L and MelanA 26-35-C A/L: Asolution of 2 ml of 3.06 mg/ml Qb VLPs in 20 mM Hepes, pH 7.2 wasreacted for 30 minutes with 18.4 μl of a solution of 50 mM SMPH(succinimidyl-6-(β-maleimidopropionoamido hexanoate, Pierce) in DMSO at25° C. on a rocking shaker. The reaction solution was subsequentlydialyzed twice for 2 hours against 2 L of 20 mM Hepes, pH 7.2 at 4° C. 2ml of the dialyzed reaction mixture was then reacted with 18.4 μl of 50mM peptide stock solution (in DMSO) for two hours at 25° C. on a rockingshaker. The reaction mixture was subsequently dialyzed 2×2 hours against2 liters of 20 mM Hepes, pH 7.2 at 4° C. The coupled products were namedQb-MelanA 16-35 (SEQ ID NO: 54), Qb-MelanA 16-35 A/L (SEQ ID NO: 55) andQb-MelanA 26-35-C A/L (SEQ ID NO: 60). For MelanA 26-35: A solution of 2ml of 3.06 mg/ml Qb capsid protein in 20 mM Hepes, pH 7.2 was reactedfor 30 minutes with 75.3 μl of a solution of 50 mM SMPH in DMSO at 25°C. on a rocking shaker. The reaction solution was subsequently dialyzedtwice for 2 hours against 2 L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml ofthe dialyzed reaction mixture was then reacted with 37.7 μl of 50 mMpeptide stock solution (in DMSO) for 4 hours at 25° C. on a rockingshaker. The reaction mixture was subsequently dialyzed 2×2 hours against2 liters of 20 mM Hepes, pH 7.2 at 4° C. The coupled product was namedQb-MelanA 26-35.

For MelanA 26-35 A/L (SEQ ID NO: 57): A solution of 2 ml of 3.06 mg/mlQb VLPs in 20 mM Hepes, pH 7.2 was reacted for 30 minutes with 37.7 μlof a solution of 50 mM SMPH in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml of the dialyzed reaction mixturewas then reacted with 18.4 μl of 50 mM peptide stock solution (in DMSO)for 4 hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.2at 4° C. The coupled product was named Qb-MelanA 26-35 A/L.

For MelanA 20-40 A/L (SEQ ID NO: 58): A solution of 2 ml of 3.06 mg/mlQb VLPs in 20 mM Hepes, pH 7.2 was reacted for 30 minutes with 18.4 μlof a solution of 50 mM SMPH in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml of the dialyzed reaction mixturewas then reacted with 184 μl of 5 mM peptide stock solution (in DMSO)for 4 hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.2at 4° C. The coupled product was named Qb-MelanA 20-40 A/L.

For MelanA 26-40 A/L (SEQ ID NO: 59): A solution of 2 ml of 3.06 mg/mlQb VLPs in 20 mM Hepes, pH 7.2 was reacted for 30 minutes with 37.7 μlof a solution of 50 mM SMPH in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml of the dialyzed reaction mixturewas then reacted with 184 μl of 5 mM peptide stock solution (in DMSO)for 4 hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.2at 4° C. The coupled product was named Qb-MelanA 26-40 A/L.

Coupling efficiency was checked by SDS-PAGE analysis. FIG. 1 shows theSDS-PAGE analysis of Qb-MelanA VLPs. MelanA-peptides were coupled to QbVLPs. The final products were mixed with sample buffer and separatedunder reduced conditions on 16% Novex® Tris-Glycine gels for 1.5 hoursat 125 V. The separated proteins were stained by soaking the gel inCoomassie blue solution. Background staining was removed by washing thegel in 50% methanol, 8% acetic acid. The Molecular weight marker (P77085, New England BioLabs, Beverly, USA) was used as reference forQb-MelanA migration velocity (lane 1). 14 μg of either Qb alone (lane 2)or Qb derivatized with SMPH (lane 3) were loaded for comparison with 8μg of each final product: Qb-MelanA 16-35 (lane 4), Qb-MelanA 16-35 A/L(lane 5), Qb-MelanA 26-35 (lane 6) and Qb-MelanA 26-35 A/L (lane7).

The MelanA 16-35 A/L peptide contains the cytotoxic T lymphocyte (CTL)epitope MelanA 26-35 and Qb-MelanA 16-35 A/L was further studied for itsimmunogenicity in vitro and in vivo.

EXAMPLE 21

Qβ MelanA 16-35 A/L VLPs are Processed by Dendritic Cells and they arePresented in Vitro to T Cells

Generation of MelanA-Specific T Cells in Vitro

In order to assess the immunogenicity of Qb-MelanA 16-35 A/L vaccine,MelanA-specific T cells were generated in vitro. Immaturemonocyte-derived dendritic cells (DC) from HLA-A2 healthy volunteerswere generated, as previously described (Salusto, F. et al. 1994, J.Exp. Med.179:1109). DC (0.5×106/well) were pulsed either mock or with 40μg/ml Qb-MelanA 16-35 A/L for 2 h at 37° C. To increase the frequency ofMelanA-specific CTL precursors, autologous CD8 T cells were isolatedfrom PBMC by magnetic sorting (Miltenyi Biotech). 1×106/well CD8 T cellswere added to antigen-pulsed DC. IL-2 was supplemented in the cellculture at day 3 at a concentration of 10 U/ml and was increased up to50 U/ml till day 12.

The expansion of MelanA-specific CD8 T cells in the cell lines wasassessed by staining with phycoerythrin (PE)-labelled tetramers made ofHLA-A2 loaded with MelanA 26-35 peptide (HLA-A2-MelanA-PE), aspreviously described (Romero, P. et all, 1998, J. Exp. Med. 1998,188:1641). Cells were labelled for 1 h at room temperature withHLA-A2-MelanA 26-35-PE, then anti-CD8-FITC antibody (BD PharMingen, SanJose, USA) was added for 30 min on ice. After washing, cells wereanalysed on a FACS Calibur using CellQuest software. Cells were acquiredin the forward scatter and side scatter and the lymphocytes were gated.From this lymphocyte population, only CD8 positive T cells were selectedfor further analyses. The amount of MelanA-specific CD8+ T cells wascalculated as percentage of HLA-A2-MelanA positive cells out of CD8+lymphocytes. Qb-MelanA 16-35 A/L vaccine induced proliferation of MelanA26-35 A/L-specific CTL (9.7% of CD8 T cells), demonstrating that thevaccine is efficiently taken up, processed to the CTL epitope (MelanA26-35 A/L) and presented by human DC to T cells.

MelanA CTL were enriched by FACS sorting (FACSVantage, Becton Dickenson)and cloned by limiting dilutions, as described previously (Knuth, A.1989, PNAS, 86:2804). CTL clones were selected for positive stainingwith HLA-A2-MelanA-PE tetramers. CTL were periodically restimulated byphitohaemaglutinin-activated heterologous irradiated PBMC.

Assessment of Antigen Recognition by CTL Clones

51Cr release assay was performed to analyze the functional activity ofMelanA CTL clones. APC were incubated for 1.5 h with 10-6M MelanA 26-35A/L peptide or 10-6M influenza M1 peptide as a negative control. APCwere incubated with Cr51 to measure the unspecific uptake ofradionucleotides at the time of peptide pulsing. After extensive washingto remove residual antigen and radioactivity, 104/well APC wereincubated with varying numbers of MelanA-specific T cells for 5 h. Thesupernatants were collected and the specific lysis of MelanA-presentingAPC by MelanA-specific T cells was calculated following the equation:% Specific lysis=((cpm experimental−cpm spontaneous)/(cpm maximum−cpmspontaneous))×100,where cpm experimental are the radioactive counts measured in theexperimental sample, cpm spontaneous are the counts obtained from APCpulsed with 51Cr without adding T cells and cpm maximum are the countsobtained from 51Cr-pulsed APC lysed with 1% NP-40.

APC loaded with MelanA 26-35 A/L but not with the irrelevant M1 peptidewere efficiently lysed by CTL (70-85% specific lysis), which confirmsthe antigenic specificity of CTL clones.

EXAMPLE 22

Capacity of Immunostimulatory Sequences (ISS) to Activate Human Cells InVitro

In order to select for the optimal ISS to be loaded in Qb-MelanAvaccine, series of CpG with different number of flanking Gs or doublestranded RNA, such as poly (I:C) were tested for their ability toupregulate CD69 on human CD8 T cells and to induce secretion of IFNalpha and IL-12 in human PBMC.

Human PBMC were isolated from buffy coats and treated with the indicatedISS in RPMI medium containing 10% FCS for 18 h. IFN alpha in thesupernatants was measured by ELISA, using an antibody set provided byPBL Biomedical Laboratories. PBMC were stained with mouse anti-humanCD8-FITC, mouse anti-human CD19-PE and anti-human CD69-APC and analyzedby flow cytometry. G10-PO was the most active ISS in inducing IFN alphasecretion (FIG. 2A). G9-9 and G8-8 were a bit less active than G10-PO,although they induced high levels of IFN alpha secretion. Decreasing thenumber of flanking Gs in the other oligonucleotides resulted in lowerIFN alpha secretion. Poly (I:C)-treated PBMC did not release any IFNalpha, although poly (I:C)-treated T and B cells from PBMC upregulatedCD69 (FIG. 2B). Poly (I:C) also induced IL-12 secretion from PBMC andmonocyte-derived DC.

Treatment of PBMC with G10-PO, G9-9 and G8-8 upregulated CD69 on thecell membrane of CD8 T cells to a nearly similar extend. Decreasing thenumber of flanking Gs (below 7) in the other oligonucleotides reducedtheir activity to induce secretion of IFN alpha (FIG. 2A) and toupregulate CD69 on T cells (FIG. 2B). These data show that G10-PO, G9-9and G8-8 have comparable high activity on human cells, therefore theycan be used as ISS in Qb-MelanA vaccine.

EXAMPLE 23

In Vitro Expansion of MelanA-Specific T Cells is Increased by G10-PO

The ability of Qb-MelanA VLPs to induce in vitro proliferation of MelanAspecific CTL was tested in the presence or absence of G10-PO. Immaturehuman monocyte-derived DC (0.5×106/well) were pulsed with eitherQb-MelanA 16-35 A/L or MelanA 26-35 A/L peptide or mock as in Example21. Human monocyte-derived DC are toll-like receptor-9 (TLR-9)-negativeand therefore they do not respond to CpG. B cells and plasmacytoid DCpresent in human PBMC are TLR-9 positive and respond to CpG treatmentwith production of cytokines (IFN alpha, IL-12).To investigate the roleof G10-PO on the antigen-presenting capacity of monocyte-derived DC,antigen-pulsed DC were washed and incubated with 1×106 autologous PBMCin the presence or absence of 2 μM G10-PO for 2 h. Autologous CD8 Tcells (1×106), isolated by magnetic sorting were added to APC andincubated for 12 days using the cell culture conditions, described inExample 21. MelanA-specific CTL were detected by HLA-A2-MelanA-PEtetramer staining and flow cytometry analysis. Adding G10-PO to theQb-MelanA-pulsed DC increased the frequency of specific CTL (from 10% to14%), which indicates that the cytokine milieu, created by CpGstimulation is favourable for CTL expansion in vitro.

EXAMPLE 24

Qbx33 VLPs Loaded with G3-6, G6, G10-PO or Poly (I:C) Induces Protectionagainst p33-Recombinant Vaccinia Virus Challenge

B6 mice were subcutaneously immunized with Qbx33 alone or loaded withG3-6 or G6 or poly (I:C) (see Examples 12 and 14). Eight days later,mice were challenged with 1.5×106 pfu of recombinant Vaccinia virus,expressing the LCMV-p33 antigen. After 4 days, mice were sacrificed andthe viral titers in ovaries were measured as previously described(Bachmann et al, Eur. J. Imunol. 1994, 24:2228). As depicted in FIG. 3,all mice receiving the Qbx33 vaccine loaded with either G3-6 or G6 orpoly (I:C) were protected from viral challenge. In contrast, naïve miceand mice immunized with Qbx33 alone did not eliminate the virus from theovaries. These data demonstrate that VLP alone is not sufficient toinduce protective CTL immune response, whereas VLP loaded with CpG orpoly (I:C) are very efficient in priming naïve CTL.

Similarly, immunization of mice with Qbx33 loaded with G10-PO waspriming p33-specific CTL (6.2%±1.4% vs 0.2%±0.1% in naïve mice), as wellas inducing protection from recombinant Vaccinia virus challenge.

EXAMPLE 25

Qβ⁻ MelanA 16-35 A/L VLPs are processed and presented by the human MHCclass I allele HLA-A0201 and induces expansion of functionalMelanA-specific CD8+T cells in HLA-A2 transgenic mice

HHD mice express a chimeric monochain class I molecule with a humanβ2-microglobulin covalently linked to the N-terminus of A2 α1 and α2domains fused with Db α3 domain (Firat, H. et al 1999, Eur.J.Immunol.,29:3112). The HLA-A2 transgene expression in these mice allowsinvestigating the capacity of Qβ MelanA 16-35 A/L VLPs to be processedand presented as the CTL epitope MelanA 26-35 and to prime CTL in vivo.Furthermore, the effect of adjuvants, as ISS can be studied in vivo.

HHD mice were either left untreated or immunized by injectingsubcutaneously 100 μg Qb-MelanA 16-35 A/L or Qb-pIC-MelanA 16-35 A/L.Eight days later spleenocytes were isolated, resuspended in FACS buffer(PBS, 2% FCS, 5 mM EDTA, pH 8.2) and stained with HLA-A2-MelanA-PElabelled tetramers for 30 min at room temperature. In a second step, ratanti-mouse CD8-APC (BD PharMingen, San Jose, USA) and anti mouseMel14-FITC (BD PharMingen, San Jose, USA) were added for 30 min at 4° C.After washing, erythrocytes were lysed with BD-Lyzing Solution (BDBiosciences, San Jose, USA) for 10 min at room temperature. Finally, thespleen cells were analysed on a FACS Calibur using CellQuest software.First of all, the cells were acquired in the forward scatter and sidescatter and the lymphocytes were gated. From this lymphocyte population,only the CD8 positive T cells were selected for further analyses. TheHLA-A2-MelanA-PE and Mel 14-FITC labelled cells were measured with theFL2 and FL1 detector, respectively. The amount of MelanA-specific,activated CD8+ T cells was calculated as percent HLA-A2-MelanA positive,Mel14 negative cells on total CD8+ lymphocytes.

Flow cytometry analysis showed that Qb-pIC-MelanA 16-35 A/L induced asurprisingly high expansion of MelanA-specific activated CD8+Mel14- Tcells (2.43% and 0.73%), which was higher compared to untreated animals(0.22% and 0.37%). It should be noted that the capacity of the vaccineincreased significantly only when Qb-MelanA was loaded with poly (I:C).

The human HLA-A2-MelanA tetramer as used above does not bind veryefficiently to mouse MelanA-specific T cells, as the protein ischimeric. Therefore we could assume a much higher degree of antigenspecific T cells in these mice.

In a similar experiment we analysed the efficiency of Qb-G10-MelanA16-35 A/L to prime CTL in vivo with vaccination with the peptide mixedwith CpG and IFA. HHD mice were immunized either with 200 μgQβ-G10-MelanA 16-35 A/L or with 50 μg MelanA 16-35 A/L mixed with 20nmol CpG and Incomplete Freud's Adjuvant (IFA) or left untreated. Eightdays later viable lymphocytes were isolated from the spleens and stainedfor MelanA-specific CD8+T cells. Staining was performed in FACS bufferfor 1.5 h at 37° C. with a PE-labelled tetramer specific for thechimeric HLA-A2α1α2Kbα3 MHC class I molecule loaded with the MelanA26-35 peptid. In a second step, rat anti-mouse CD8-APC (BD PharMingen,San Jose, USA) and anti mouse Mell4-FITC (BD PharMingen, San Jose, USA)was added for 30 min at 4° C. Finally, the spleen cells were analysed ona FACS Calibur using CellQuest software.

Flow cytometry analysis showed that Qβ-G10-MelanA 16-35 A/L inducedexpansion of MelanA-specific activated CD8⁺Mel14⁻T cells (18.2%) whichwas higher compared to untreated animals (2%) or animal receivingequimolar amounts of MelanA A/L 16-35 mixed with 20 nmol CpG and IFA(2.0%). It should be noted that the capacity of the vaccine increasedonly when Qb-MelanA was loaded with G10-PO.

In a similar experimental setting, immunization of HHD mice withQb-MelanA 16-35 A/L or Qb-MelanA 26-35 A/L loaded with G8-8 or G10 POinduces expansion of HLA-A2-MelanA—positive and Mell4 negative CD8 Tcells.

Taken together these findings demonstrate the ability of ISS loadedQb-peptide vaccines to very efficiently prime CTL against foreign andself antigens.

EXAMPLE 26

Qbx33 Loaded with CpG can be used in Homologous as Well in HeterologousPrime-Boost Regimen for the Induction of a Long Lasting Memory CD8+ TCell Response

Mice were immunized with 150 ug Qbx33/NKCpG and 8 days later thefrequencies of p33-specific T cells increased from 0.4%±0.2% in naïvemice to 7.5%±2.2% in immunized animals as measured with antigen.specificMHC/peptide tetramers. 20 days later the peptide specific CD8+ Tpopulation dropped down to 1.6%±0.7%. A second-immunization of thesemice 30 days after the first immunisation with 150 ug Qbx33/NKPS couldboost the memory T cell response to up to 8.4%±1.9% specific T cells.This response dropped slowly down but could be boosted again 4 monthsafter the first boost with 150 ug Qbx33/NKPS reaching T cell levels of23.8%±5.2%.

When 3 mice were primed with 50 ug p33 peptide mixed with 20 nmol NKPSand IFA only 0.6%± 0.4% specific CD8+ T cells could be induced until day8 post-immunisation. Nevertheless, this low response could be boostedefficiently 7 weeks later with Qbx33/NKPS to levels of 28.5%±9.8%.

Immunisation with 1×10 exp 6 plaque forming units of recombinantvaccinia virus expressing the p33-peptide could hardly induce any T cellresponse (1.1%±0.5%) but was boosted very efficiently boosted 6 monthslater with 150 ug Qbx33/NKPS to T cells levels of 28.1± 4.2%.

These results show, that Qb loaded with CpG very efficiently boosts anypre-existing T cell response in heterologous as well as homologous primeboost regimens. It should be noted, that Qb/NKPS can even boost a veryinefficiently primed T cell response with peptides or recombinantviruses. In addition, when a strong T cell response was established withQbx33/NKPS we were able to boost this response using an immunologicallyeffective amount of a heterologous vaccine such as the p33 peptidealone, recombinant virus expressing p33, or p33 fused or coupled to aVLP. In the latter, the used VLP is not a VLP derived from RNA phage Qbbut e.g. HBcAg or VLP derived from AP205.

1. A composition comprising: (a) a virus-like particle; (b) at least oneimmunostimulatory substance; (i) wherein said immunostimulatorysubstance is an immunostimulatory nucleic acid; and (ii) wherein saidimmunostimulatory substance is packaged into said virus-like particle;and (c) at least one antigen or antigenic determinant; (i) wherein saidantigen or antigenic determinant is bound to said virus-like particle;and (ii) wherein said antigen or antigenic determinant comprises a humanmelanoma MelanA peptide analogue that comprises an amino acid sequencederived from the amino acid sequence of SEQ ID NO:78 or SEQ ID NO:79 byalteration of one or two amino acid(s) or amino acid derivative(s) insaid amino acid sequence, wherein said alteration comprises an aminoacid substitution, deletion or insertion or a combination thereof. 2.The composition of claim 1, wherein said antigen or antigenicdeterminant is bound to said virus-like particle by at least onenonpeptide covalent bond.
 3. The composition of claim 1, wherein saidhuman melanoma MelanA peptide analogue is characterized by one or twoamino acid substitutions with respect to the normal MelanA peptide. 4.The composition of claim 1, wherein said human melanoma MelanA peptideanalogue comprises an amino acid sequence selected from the groupconsisting of: (a) LAGIGILTV (SEQ ID NO:84); (b) MAGIGILTV (SEQ IDNO:85); (c) EAMGIGILTV (SEQ ID NO: 86); (d) ELAGIGILTV (SEQ ID NO: 50);(e) EMAGIGILTV (SEQ ID NO: 87); (f) YAAGIGILTV (SEQ ID NO: 88); and (g)FAAGIGILTV (SEQ ID NO: 89).
 5. The composition of claim 1, wherein saidhuman melanoma MelanA peptide analogue comprises the sequence ELAGIGILTV(SEQ ID NO:50).
 6. The composition of claim 1, wherein said virus-likeparticle comprises at least one first attachment site and wherein saidantigen or antigenic determinant further comprises at least one secondattachment site being selected from the group consisting of: (a) anattachment site not naturally occurring with said antigen or antigenicdeterminant; and (b) an attachment site naturally occurring with saidantigen or antigenic determinant; and wherein said binding of saidantigen or antigenic determinant to said virus-like particle is effectedthrough association between said first attachment site and said secondattachment site and wherein said antigen or antigenic determinant andsaid virus-like particle interact through said association to form anordered and repetitive antigen array.
 7. The composition of claim 6,wherein said first attachment site comprises an amino group.
 8. Thecomposition of claim 6, wherein said second attachment site comprises asulfhydryl group.
 9. The composition of claim 6, wherein said firstattachment site is an amino group and said second attachment site is asulfhydryl group.
 10. The composition of claim 6, wherein said humanmelanoma MelanA peptide analogue with said second attachment sitecomprises an amino acid sequence selected from the group consisting of:(a) CGHGHSYTTAEELAGIGILTV (SEQ ID NO:55); (b) CGGELAGIGILTV (SEQ IDNO:57); (c) CSYTTAEELAGIGILTVILGVL (SEQ ID NO:58); (d)CGGELAGIGILTVILGVL (SEQ ID NO:59); (e) ELAGIGILTVGGC (SEQ ID NO:60); (f)CSPKSLELAGIGILTV (SEQ ID NO:92); and (g) ELAGIGILTVILGVLGGC (SEQ IDNO:93).
 11. The composition of claim 6, wherein said human melanomaMelanA peptide analogue with said second attachment site comprises theamino acid sequence CGHGHSYTTAEELAGIGILTV (SEQ ID NO:55).
 12. Thecomposition of claim 1, wherein said virus-like particle is arecombinant virus-like particle, wherein said virus like particlecomprises recombinant proteins selected from the group consisting of:(a) recombinant proteins of Hepatitis B virus; (b) recombinant proteinsof measles virus; (c) recombinant proteins of Sindbis virus; (d)recombinant proteins of Rotavirus; (e) recombinant proteins ofFoot-and-Mouth-Disease virus; (f) recombinant proteins of Retrovirus;(g) recombinant proteins of Norwalk virus; (h) recombinant proteins ofhuman Papilloma virus; (i) recombinant proteins of BK virus; (j)recombinant proteins of bacteriophages; (k) recombinant proteins ofRNA-phages; (l) recombinant proteins of Ty; and (m) fragments of any ofthe recombinant proteins from (a) to (l).
 13. The composition of claim1, wherein said virus-like particle comprises recombinant proteins, orfragments thereof, of a RNA-phage, wherein said RNA-phage is selectedfrom the group consisting of: (a) bacteriophage Qβ; (b) bacteriophageR17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP;(f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i)bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7; and (l)bacteriophage AP205.
 14. The composition of claim 1, wherein saidvirus-like particle comprises recombinant proteins, or fragmentsthereof, of bacteriophage Qβ or bacteriophage AP205.
 15. The compositionof claim 1, wherein said immunostimulatory substance is a toll-likereceptor activating substance, and wherein said immunostimulatorynucleic acid is selected from the group consisting of: (a) ribonucleicacids; (b) deoxyribonucleic acids; (c) chimeric nucleic acids; and (d)mixtures of at least one nucleic acid of (a), (b) and/or (c).
 16. Thecomposition of claim 15, wherein said deoxyribonucleic acid is anunmethylated CpG-containing oligonucleotides.
 17. The composition ofclaim 1, wherein said immunostimulatory substance is an unmethylatedCpG-containing oligonucleotide.
 18. The composition of claim 17, whereinsaid unmethylated CpG-containing oligonucleotide comprises the sequence:5′ X1X2CGX3X4 3′ and wherein X1, X2, X3, and X4 are any nucleotide; andwherein at least one of said nucleotide X1, X2, X3, and X4 has aphosphate backbone modification.
 19. The composition of claim 17,wherein said unmethylated CpG-containing oligonucleotide comprises apalindromic sequence.
 20. The composition of claim 17, wherein saidunmethylated CpG-containing oligonucleotide consists of the sequenceGGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:4).
 21. The composition ofclaim 19, wherein said palindromic sequence comprises GACGATCGTC (SEQ IDNO:1).
 22. A method for enhancing an immune response against an antigenin an animal comprising introducing the composition of claim 1 into saidanimal, wherein an enhanced immune response against said antigen isproduced in said animal.
 23. The method of claim 22, wherein said immuneresponse is an enhanced B cell response or an enhanced T cell response.24. The method of claim 22, wherein said animal is a mammal.
 25. Themethod of claim 22, wherein said composition is introduced into saidanimal subcutaneously, intramuscularly, intravenously, intranasally ordirectly into the lymph node.
 26. An immunogenic composition comprisingan immunologically effective amount of the composition of claim 1together with a pharmaceutically acceptable diluent, carrier orexcipient.
 27. A method of inmmnizing or treating an animal comprisingadministering to said animal an immunologically effective amount of theimmunogenic composition of claim
 26. 28. The method of claim 27, whereinsaid animal is a mammal.
 29. A method of immunizing or treating ananimal comprising the steps of priming a T cell response in said animal,and boosting a T cell response in said animal, wherein said priming orsaid boosting is effected by administering an immunologically effectiveamount of the immunogenic composition of claim
 26. 30. The method ofclaim 29, wherein said priming and said boosting is effected byadministering an immunologically effective amount of said immunogeniccomposition.
 31. The method of claim 23, wherein said T cell response isa CTL response or a Th cell response.
 32. The method of claim 31,wherein said Th cell response is a Th1 cell response.
 33. The method ofclaim 24, wherein said mammal is a human.
 34. The immunogeniccomposition of claim 26, wherein said immunogenic composition furthercomprises an adjuvant.
 35. The method of claim 28, wherein said mammalis a human.
 36. The composition of claim 1, wherein said antigen orantigenic determinant consists of a human melanoma MelanA peptideanalogue comprising an amino acid sequence derived from the amino acidsequence of SEQ ID NO:78 or SEQ ID NO:79 by alteration of one or twoamino acid(s) or amino acid derivative(s) in said amino acid sequence,wherein said alteration comprises an amino acid substitution, deletionor insertion or a combination thereof.
 37. The composition of claim 1,wherein said antigen or antigenic determinant consists of a humanmelanoma MelanA peptide analogue consisting of an amino acid sequencederived from the amino acid sequence of SEQ ID NO:78 or SEQ ID NO:79 byalteration of one or two amino acid(s) or amino acid derivative(s) insaid amino acid sequence, wherein said alteration comprise an amino acidsubstitution, deletion or insertion or a combination thereof.
 38. Thecomposition of claim 1, wherein said amino acid substitution, deletionor insertion or a combination thereof is at position 1, 2, 3 or 10 ofSEQ ID NO:78 or a combination thereof or at position 1, 2 or 9 of SEQ IDNO:79 or a combination thereof.
 39. The composition of claim 1, whereinsaid human melanoma MelanA peptide analogue comprises an amino acidsequence derived from the amino acid sequence of SEQ ID NO:78 or SEQ IDNO:79 by substitution of one amino acid.
 40. The composition of claim39, wherein said substitution is at position 1, 2, 3 or 10 of SEQ IDNO:78 or a combination thereof or at position 1, 2 or 9 of SEQ ID NO:79or a combination thereof.
 41. The composition of claim 1, wherein saidhuman melanoma MelanA peptide analogue consists of the sequenceELAGIGILTV (SEQ ID NO:50).
 42. The composition of claim 1, wherein saidvirus-like particle comprises at least one first attachment site andwherein said antigen or antigenic determinant further comprises at leastone second attachment site selected from the group consisting of: (a) anattachment site not naturally occurring with said antigen or antigenicdeterminant; and (b) an attachment site naturally occurring with saidantigen or antigenic determinant; wherein said binding of said antigenor antigenic determinant to said virus-like particle is effected throughassociation between said first attachment site and said secondattachment site.
 43. The composition of claim 42, wherein said antigenor antigenic determinant and said virus-like particle interact throughsaid association to form an ordered and repetitive antigen array. 44.The composition of claim 1, wherein said virus-like particle is avirus-like particle of an RNA-phage.
 45. The composition of claim 1,wherein said virus-like particle is a virus-like particle of RNA-phageQβ.
 46. The composition of claim 1, wherein said virus-like particlecomprises recombinant proteins of bacteriophage Qβ.
 47. The compositionof claim 1, wherein said virus-like particle comprises recombinantproteins of bacteriophage Qβ, wherein said recombinant proteins compriseSEQ ID NO:10.
 48. The composition of claim 1, wherein said virus-likeparticle comprises recombinant proteins of bacteriophage Qβ, whereinsaid recombinant proteins consist of SEQ ID NO:10.
 49. The compositionof claim 15, wherein said ribonucleic acids arepolyinosinic-polycytidylic acid double-stranded RNA (poly-(I:C)). 50.The composition of claim 1, wherein said immunostimulatory nucleic acidis not accessible to DNAse hydrolysis.
 51. The composition of claim 45,wherein said unmethylated CpG-containing oligonucleotide consists of thesequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:41).
 52. Thecomposition of claim 2, wherein said virus-like particle is a virus-likeparticle of RNA-phage Qβ.
 53. The composition of claim 52, wherein saidvirus-like particle comprises recombinant proteins of bacteriophage Qβ,and wherein said human melanoma MelanA peptide analogue consists of thesequence ELAGIGILTV (SEQ ID NO:50).
 54. The composition of claim 53,wherein said virus-like particle comprises at least one first attachmentsite and wherein said antigen or antigenic determinant further comprisesat least one second attachment site selected from the group consistingof: (a) an attachment site not naturally occurring with said antigen orantigenic determinant; and ( b) an attachment site naturally occurringwith said antigen or antigenic determinant; wherein said binding of saidantigen or antigenic determinant to said virus-like particle is effectedthrough association between said first attachment site and said secondattachment site, and wherein said antigen or antigenic determinant andsaid virus-like particle interact through said association to form anordered and repetitive antigen array.
 55. The composition of claim 54,wherein said first attachment site is an amino group and said secondattachment site is a sulfhydryl group.
 56. The composition of claim 55,wherein said immunostimulatory substance is an unmethylatedCpG-containing oligonucleotide, and wherein said unmethylatedCpG-containing oligonucleotide consists of the sequenceGGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:41).
 57. The composition ofclaim 56, wherein said virus-like particle comprises recombinantproteins of bacteriophage Qβ, wherein said recombinant proteins consistof SEQ ID NO:10.
 58. The composition of claim 57, wherein said humanmelanoma MelanA peptide analogue with said second attachment siteconsists of the amino acid sequence CGHGHSYTTAEELAGIGILTV (SEQ IDNO:55).